New Aspects of Septin Assembly and Cell Cycle Control in Multinucleated A

New aspects of septin assembly and cell cycle control in multinucleated A. gossypii

Inauguraldissertation

zur

Erlangung der Würde eines Doktors der Philosophie

vorgelegt der

Philosophisch-Naturwissenschaftlichen Fakultät

der Universität Basel

von

Hanspeter Helfer aus Selzach, SO

Basel, 2007

Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von

Prof. Dr. Peter Philippsen, Prof. Dr. Anne Spang und Prof. Dr. Jean Pieters

Basel, 6. Juni 2006

Prof. Dr. Hans-Jakob Wirz

Dekan

Table of Contents

Summary 5

General Introduction 6

Chapter I A conserved cell cycle control module leading to CDK tyrosine phosphorylation functions in the A. gossypii starvation response 9

Introduction 9

Results 11 - Phosphorylation of Cdc28-Y18 is a candidate for regulation of the nuclear cycle 11 - Minimal AgCdc28-Y18 phosphorylation under ideal growth conditions 11 - AgCdc28p is phosphorylated on tyrosine 18 when hyphae are starved for nutrients 11 - The nuclear cycle is delayed in specific stages of division during starvation 12 - Homologues of the yeast morphogenesis checkpoint components in A. gossypii 12 - AgSwe1p is responsible for AgCdc28Y18 phosphorylation 13 - Only minimal role of morphogenesis checkpoint homologues in regulating nuclear density under non-starving conditions 13 - Starvation induced nuclear cycle delay depends on AgSwe1p 13 - Mitosis are concentrated at sites of branch formation 14 - Table 1. Homologues of the yeast MC components in A. gossypii 16

Discussion 17

Chapter II Septins – cytokinesis factors in the absence of cell division in A. gossypii 20

Introduction 20

Results 22 - Septins are conserved in two organisms of most diverse morphology 22 - Continuous AgSep7p-GFP ring at the neck of budding yeast 22 - Intermitted hyphal AgSep7p-GFP rings in A. gossypii 22 - Variety of septin organizations 23 - Chronological order of septin localization 23 - One ring to rule them all 23 - A. gossypii septins are not essential but required for efficient growth and proper morphogenesis 24 - Cortical barrier? 24 - Separating the “twins” (AgCDC11A/B) 25

Discussion 26

TABLE OF CONTENTS 3 Materials and Methods 29

- A. gossypii methods, media and growth conditions 29 - Plasmid and strain construction 29 - Protein extraction and Western blotting 31 - Fluorescence stainings, image acquisition and processing 31 - Table 2. A. gossypii strains used in this study 33 - Table 3. Plasmids used in this study 34 - Table 4. Oligonucleotide primers used in this study 35

Appendix 37

I Verification PCRs

II Plasmid maps

III Sequence alignments

References 38

Acknowledgments 48

Curriculum vitae 49

TABLE OF CONTENTS 4 Summary

Chapter I Nuclei in the filamentous ascomycete Ashbya gossypii divide asynchronously and most nuclei have the potential to divide (Alberti-Segui et al., 2001; Gladfelter et al., 2006). Although cytoplasmic extension is restricted to growing tips and emerging branches, the distances between nuclei are uniform along the entire hyphal length. This implies active control of nuclear distribution and division to maintain an ideal nuclear to cytoplasmic ratio, potentially depending on environmental conditions. The question of nuclear distribution has already been addressed earlier (Alberti-Segui et al., 2001). We have investigated how the rate of mitosis is regulated in response to intra- and extracellular signals. Here we show that homologues of S. cerevisiae morphogenesis checkpoint components are involved in starvation response in A. gossypii: Phosphorylation of the cyclin dependent kinase AgCdc28p at tyrosine 18 by the protein kinase AgSwe1p is used to delay mitosis under low-nutrient conditions, leading to an increase in the average distance between nuclei. This effect is markedly reduced in Agswe1Δ or Agcdc28Y18F mutants where the CDK cannot be phosphorylated. Overexpression of AgSWE1 leads to decreased nuclear density even under non-starving conditions. Addition of rapamycin mimics starvation response, suggesting that AgSwe1p may be under control of AgTor1/2p.

In unperturbed budding yeast cells, ScSwe1p is recruited to the septin ring at the mother-bud neck where it is phosphorylated and subsequently degraded. We have speculated that the septins in A. gossypii could serve as spatial markers to locally inactivate AgSwe1p and increase nuclear division rate in areas of growth. Time-lapse analysis has revealed that mitoses in wild type are most common near branching points. Interestingly, AgSep7p-GFP localizes to branching points and septin deletion mutants show random distribution of mitoses. We propose a model in which AgSwe1p may regulate mitosis in response to cell intrinsic morphogenesis cues and external nutrient availability in multinucleated cells.

Chapter II Septins are evolutionary conserved proteins with essential functions in cytokinesis, and more subtle roles throughout the cell cycle. Much of our knowledge about septins originates from studies with S. cerevisiae, where they form a ring-like protein scaffold at the mother-bud neck. We have asked what functions the septins may hold in an organism that does not complete cytokinesis prior to sporulation. Interestingly, all budding yeast septins are conserved in A. gossypii and one is even duplicated (S. Brachat, personal communication; Dietrich et al., 2004). In vivo studies of AgSep7p-GFP have revealed that septins assemble into discontinuous hyphal rings close to growing tips and sites of branch formation and into asymmetric structures at the base of branching points. Rings are made of filaments which are long and diffuse close to growing tips and short and compact further away from the tip. During septum formation, the septin ring splits into two to form a double ring.

Agcdc3Δ, Agcdc10Δ, and Agcdc12Δ mutants display aberrant morphology and are defective for actin- ring formation, chitin-ring formation, and sporulation. Due to the lack of septa, septin deletion mutants are highly sensitive, and lesion of a single hypha can have catastrophic consequences for a young mycelium. Strains lacking AgCDC11A show morphological defects comparable with other septin deletion mutants, but actin- and chitin-ring formation are not disabled. Deletion of AgCDC11B results in no detectable phenotype under standard laboratory conditions.

SUMMARY 5 General Introduction

Filamentous fungi differ in many striking ways genetic functional analysis (Steiner et al., 1995; from common single-celled eukaryotes. The Altmann-Johl and Philippsen, 1996; Mohr, PhD mycelium formed by hyphal growth is a dense, Thesis, 1997; Wendland et al., 2000; Wendland complex network of branched filaments, whose and Philippsen, 2000; Dietrich et al., 2004). size is only limited by external conditions such as availability of resources, environmental The A. gossypii life cycle starts with the only stresses, and competing organisms. Mitosis are known phase of isotropic growth in wild type: either synchronized (all nuclei divide germination of the haploid spore to form a germ simultaneously), or they occur in an bubble. This is followed by apical growth, asynchronous manner (Nygaard et al., 1960; extending two germ tubes in succession on Clutterbuck, 1970). In either way, nuclear opposing sites of the germ bubble. More axes of division is not followed by cytokinesis, leading to polarity are established with lateral branch multinucleated hyphae which share one common formation in young mycelium. Maturation is cytoplasm. Thus, although by appearance such characterized by apical branching and a dramatic mycelia certainly do not correspond to the increase of growth speed (up to 200 μm/h at common perception of a cell, a mycelium formed 30°C, (Knechtle et al., 2003)), which enables it to by true hyphal growth has to be considered as cover an 8 cm-Petri-dish of full medium in about one single, multinucleated cell. Filamentous 7 days. Sporulation is thought to be induced by growth enables these organisms to rapidly cover nutrient deprivation, leading to contraction at the and exploit solid surfaces, and the polarized septa, cytokinesis and subsequent abscission of force generated by apical extension allows them sporangia which contain up to 8 haploid spores to penetrate tissue which would otherwise not be (Figure Intro.1, adopted from Brachat, PhD accessible. However, this growth mode also thesis, 2003) (Ayad-Durieux et al., 2000; bears new challenges: How can one single cell Brachat, PhD thesis, 2003; Knechtle et al., have the flexibility to react to conditions, which 2003). Hyphae are compartmentalized by septa, may greatly differ from one part of the mycelium which in young parts appear as rings that allow to the other? How can it prevent local damage transfer of nuclei (Alberti-Segui et al., 2001) and from spreading through the entire mycelium? in older parts may appear as closed discs. How are hyphal extension and branching Compartments typically contain around eight regulated to ensure optimized growth and nuclei. Nuclear pedigree analyses carried out by resource exploitation? How does such a cell Peter Philippsen and Amy Gladfelter have shown maintain an optimal protein per cytoplasm ratio, that neighboring nuclei are in different division although the cytoplasmic volume may increase cycle stages. Most nuclei have the potential to with various speed in different places of the divide, with the time between two divisions mycelium? varying greatly from 46 – 250 min (Gladfelter et al., 2006). Despite limitation of growth to tips and We sought to study some of these questions in branching sites, distances between the nuclei the filamentous Ascomycete Ashbya gossypii, a are largely uniform along the entire hyphal pathogen of cotton and citrus fruits and length, with an average distance of 4 – 5 μm. phylogenetically a close relative of S. cerevisiae This is surprising as one might expect (Ashby and Nowell, 1926; Prillinger et al., 1997; cytoplasmic expansion to cause a decrease of Wendland et al., 1999; Dietrich et al., 2004). A. nuclear density in areas of growth. The fact that gossypii is exclusively found in a filamentous, this is not the case indicates active maintenance multinucleated hyphal form. It has a very small of nuclear to cytoplasmic ratio along the hyphae. haploid genome (9.2 Mb) encoding 4718 genes, How may such a regulation be achieved? C. which shows a high degree of synteny Alberti-Segui has shown that deletion of the (conservation of gene order between different microtubule-based dynein motor AgDhc1p leads species) and homology with the S. cerevisiae to clustering of nuclei at the hyphal tips of A. genome. Combined with highly efficient gossypii. Thus, long range nuclear migration has homologous recombination allowing PCR-based been identified as the key element in regulating gene targeting, these features make A. gossypii nuclear distribution (Alberti-Segui et al., 2001). an attractive system to perform studies of Besides nuclear distribution, also the division regulation of filamentous growth based on rate of nuclei needs to be controlled in order to

GENERAL INTRODUCTION 6 maintain an ideal nuclear to cytoplasmic ratio. formation), the chromosome cycle (replication) This ratio may vary depending on environmental and the centrosome cycle (SPB cycle). Each of conditions. Mutants where such regulatory these cycles depends on START for initiation mechanisms are affected should display altered and each must be completed for cells to undergo nuclear densities under standard growth the metaphase to anaphase transition. cdc28 conditions. Deletion of the kinesin motor mutants have a terminal phenotype at this AgCin8p leads to a dramatic increase of the principal restriction point of the yeast cell division distance between two nuclei to about 11 μm cycle. This lead to the discovery of cyclin (Alberti-Segui, PhD thesis, 2001). However, dependant kinases (CDKs), whose changes in AgCin8p generates the pushing force of the activity are responsible for driving key cell cycle nuclear spindle and therefore has to be regarded transitions (Hartwell et al., 1974; Nasmyth and as a requirement for efficient mitosis rather than Reed, 1980). S. cerevisiae possesses at least a regulator. What do we know about regulation five CDKs, with Cdc28p being the principal of nuclear division in other organisms? regulator of the cell cycle. Cdc28p activity is regulated by nine cyclins that accumulate, for the The molecular machinery of eukaryotic cell cycle most part, at START and at G2/M that are control is most fully worked out for budding yeast regulated transcriptionally and proteolytically, Saccharomyces cerevisiae. Under conditions of several protein kinases (e.g. Sic1p, Swe1p) and limited nutrients, budding yeast cells arrest as phosphoprotein phosphatases (e.g. Mih1p), and small, unbudded cells in early G1 phase (“Gap- at least four other binding proteins. Cdc28p 1"). Addition of nutrients allows them to begin cyclins have been classified into two groups: the growing. If they achieve a critical size and no three G1 cyclins Cln1p, Cln2p, and Cln3p, and mating pheromone is present, the cells commit the six B-type (mitotic) cyclins Clb1p to Clb6p to a complete cell cycle. This execution point at (Richardson et al., 1989; Surana et al., 1991; late G1 is called START (Pringle and Hartwell, Fitch et al., 1992; Richardson et al., 1992; 1981; Tyers et al., 1992). Bud emergence Schwob and Nasmyth, 1993). Regulation of corresponds to the initiation of S phase cyclin associated Cdc28p activity is mainly (Synthesis), during which DNA replication, achieved by successive waves of cyclins, spindle pole body (SPB, fungal equivalent of the provided by a mechanism of transient expression centrosome) separation and nuclear positioning and subsequent degradation. Each wave occur. In G2 phase (Gap-2), apical growth of the stimulates degradation of the preceding cyclins bud is replaced by isotropic growth, and the and expression of the following cyclins. This SPBs migrate to opposite sides of the nucleus allows the Cyclin-CDK complex to drive the cell forming a short metaphase spindle which is through the different stages of the cell cycle. oriented in parallel to the mother-bud axis (Byers and Goetsch, 1975; Jacobs et al., 1988; Although bud formation, DNA replication and the Donaldson and Kilmartin, 1996; Carminati and centrosome cycle can proceed independently Stearns, 1997; Shaw et al., 1997). During M from each other once START has been passed, phase (Mitosis), the spindle extends at least 5- other events require tight coordination. fold, separating the sister chromatids to opposite Surveillance mechanisms termed checkpoints poles, thus leading to nuclear division (Winey are set at various stages of the cell cycle and and Byers, 1993; Snyder, 1994). With mitotic exit allow delayed processes to catch up and re- the anaphase spindle disassembles, and the cell coordiante upon perturbation (Hartwell and cycle is concluded by cytokinesis and cell Weinert, 1989). START itself can be regarded as separation (abscission) (Kilmartin and Adams, checkpoint, preventing initiation of a cell division 1984; Epp and Chant, 1997; Bi et al., 1998; cycle under bad nutritional conditions or in the Lippincott and Li, 1998b, 1998a; Vallen et al., presence of mating partners (Gallego et al., 2000). 1997). TOR (target of rapamycin) signaling plays a central role in adjusting cell growth to the Lee Hartwell spearheaded cell cycle research in nutritional environment (Barbet et al., 1996; S. cerevisiae with his screen for conditional cell Helliwell et al., 1998; Harris and Lawrence, 2003; division cycle (cdc) mutants in the 1960s and Jacinto and Hall, 2003; Wullschleger et al., 1970s (Hartwell, 1971a). Under restrictive 2006). To prevent the cells from disastrous conditions, cdc mutants arrest with characteristic segregation of incompletely replicated or cell division morphologies, called terminal damaged DNA, the “DNA damage and phenotypes. His detailed examination of these replication checkpoint” can delay the cell cycle in mutants revealed interdependence of many cell- G1 and prior to anaphase in multiple ways. The cycle events but also demonstrated that at least “morphogenesis checkpoint” prevents formation three independent cycles can be distinguished in of binucleate cells by delaying nuclear division budding yeast: the cytoplasmic cycle (bud until a bud has been formed (Lew and Reed,

GENERAL INTRODUCTION 7 1995; Sia et al., 1996; McMillan et al., 1998). The functions of S. cerevisiae septins are far Normally, the kinase Swe1p is recruited to the from being restricted to the recruitment of mother-bud neck in G2 by the concerted action morphogenesis checkpoint components. This of Hsl7p, Hsl1p and the septins. At the neck, it is conserved family of GTP-binding proteins was phosphorylated by the polo-like kinase Cdc5p, discovered through Lee Hartwell’s genetic leading to SCF-mediated ubiquitination and screening for S. cerevisiae mutants defective in subsequent degradation (Sakchaisri et al., cell-cycle progression (Hartwell, 1971b). The 2004). Inhibitory Y19 phosphorylation of Cdc28p original temperature sensitive septin mutants is reversed by the phosphatase Mih1p (Sia et al., (cdc3, cdc10, cdc11 and cdc12) form 1996; McMillan et al., 1999b). Perturbations of multinucleated cellular clusters with elongated the actin cytoskeleton delay bud formation and buds and thus were assigned essential functions therefore recruitment of neck proteins. Swe1p in cytokinesis (Cooper and Kiehart, 1996; remains stable and inhibits Cdc28p by Y19 Longtine et al., 1996; Field and Kellogg, 1999). phosphorylation. The morphogenesis checkpoint Together with a fifth septin, ScSep7p (ScShs1p), is described in detail in Chapter I: Introduction. the septins form a ring encircling the mother-bud Bipolar attachment of all the chromosomes to neck, but they also occur at the presumptive bud the mitotic spindle is monitored by the “spindle site, at the bud scar after cytokinesis and at the assembly checkpoint” prior to anaphase. This tip of the shmoo in presence of mating checkpoint is important for equal distribution of pheromones (Longtine et al., 1996; Mino et al., chromosomes to the mother and daughter cell 1998; Gladfelter et al., 2001b). The septin ring (Hoyt et al., 1991; Li and Murray, 1991; Weiss serves several functions (Kinoshita, 2003): and Winey, 1996; Amon, 1999). The “nuclear Firstly, it is a spatial landmark for bud site migration checkpoint” (also known as cytokinesis selection (Chant et al., 1995; Sanders and or spindle position checkpoint) couples exit from Herskowitz, 1996). Further, the septins form a mitosis to the correct migration of one nucleus cortical barrier that prevents membrane- into the bud (Bardin et al., 2000; Bloecher et al., associated molecules from diffusing from the bud 2000; Pereira et al., 2000). cortex into the mother cortex (Barral et al., 2000; Takizawa et al., 2000; Faty et al., 2002). They Could such a surveillance mechanism have form a scaffold to recruit molecules for the adapted to regulate the rate of mitosis in A. cytokinesis apparatus, cell-wall synthesis, and gossypii? One could say that the morphogenesis for positioning of the mitotic spindle (DeMarini et checkpoint in S. cerevisiae regulates “nuclear al., 1997; Kusch et al., 2002). Finally, they recruit density” by preventing formation of the protein kinases ScGin4p, ScKcc4p and multinucleated cells. It has been argued that not ScHsl1p, which belongs to the morphogenesis only formation of a proper neck but also checkpoint, and one component of the mitosis cytoplasmic extension of the bud could be a exit network, ScTem1p (Carroll et al., 1998; requirement before the cells are released into Jimenez et al., 1998; Longtine et al., 1998; Barral mitosis (Harvey and Kellogg, 2003). Recent et al., 1999; Lippincott et al., 2001). analyses have demonstrated that ScSwe1p is also used to delay mitosis in response to Despite profound morphological differences environmental signals such as high osmolarity between budding yeast and A. gossypii, (Clotet et al., 2006). We sought to investigate homologues to all five mitotic S. cerevisiae whether A. gossypii homologues of septins have been identified in A. gossypii, with morphogenesis checkpoint components CDC11 being duplicated (S. Brachat, personal (AgSwe1p, AgMih1p, AgHsl1p, AgHsl7p, communication; (Dietrich et al., 2004). What septins) have evolved to control nuclear division function do the septins have in an organism that rate in response to hyphal extension and does not undergo complete cytokinesis unless extracellular stimuli such as stress and for sporulation? Where do they localize in the availability of nutrients (Chapter I). The septins absence of a bud neck or a division plane? The are crucial for the recruitment and inactivation of aim of Chapter II is to shed light on these ScSwe1p in budding yeast. Thus, we further questions by studying localization of AgSep7p- investigated the possibility of morphogenesis GFP and by functional analyses of deletion checkpoint proteins providing spatial control of mutants. mitosis, with the septins functioning as cortical markers to locally trigger mitosis in areas of growth.

GENERAL INTRODUCTION 8

Figure Intro.1. A. gossypii life cycle (adopted from S. Brachat, Thesis, 2003). A. gossypii mycelium grown on solid medium gives rise to needle-shaped spores, recognizable as white fluff in the middle of the colony. Isotropic growth during germination leads to the formation of a germ bubble in the center of the spore. After a permanent switch to polarized, apical growth, two germ-tubes emerge successively on opposing sites of the germ bubble. Nuclear division occurs without subsequent cytokinesis, leading to multinucleated hyphae. New axis of polarity are established by lateral branching in young mycelium. During maturation of the mycelium, the tip extension rate is markedly increased and apical branching (tip splitting) now is the predominant form of branching. Upon growth for an extended time, the sporulation program is initiated, leading to the formation of sporangia and concluding the cycle. Chapter I

A conserved cell cycle control module leading to CDK tyrosine phosphorylation functions in the A. gossypii starvation response

Introduction

Multinucleated cells are common throughout the mycelium could spatially direct asynchronous biosphere. Filamentous, pathogenic fungi, mitosis in multinucleated hyphae. metastasizing tumor cells and the cells of the musculoskeletal and blood systems of mammals To prevent the formation of aberrant binucleate are examples of the diverse types of cells which cells in Saccharomyces cerevisae, it is crucial contain many nuclei in one cytoplasm (Mills and not to start nuclear division before a proper bud Frausto, 1997; Woodhouse et al., 1997; Su et has been formed. Therefore, mitotic entry is al., 1998; Wakefield et al., 2000; Winding et al., tightly coordinated with bud formation by the 2000; Atkins et al., 2001). In some cases these morphogenesis checkpoint (Figure I.1). Mitosis is cells are produced from specialized cell cycles in promoted by a complex of mitotic cyclin with the which nuclear division occurs without cell cyclin dependant kinase (ScCdc28p). When bud division leading to large syncytia. For fungi, formation is delayed due to defects in the actin multinucleated cells may extend over hundreds cytoskeleton or if the septin scaffold (see of meters so that different regions of a single cell Chapter II) is perturbed then the intrinsically experience dramatically different microenviron- unstable Swe1p kinase (Wee1 homologue) is ments. stabilized and translocates to the nucleus where it can inhibit Cdc28/Clb2p, through phosphory- Mitosis in multinucleated cells can occur either in lation of the tyrosine 19 residue on Cdc28p (Sia a coordinated, synchronous manner where all et al., 1996; McMillan et al., 1998; Sia et al., nuclei divide simultaneously or asynchronously 1998; McMillan et al., 1999a; McMillan et al., where individual nuclei divide independently in 1999b). This provides a G2-delay for cells to time and space. Both synchronous and asyn- recover proper morphogenesis prior to mitosis chronous patterns of mitosis are observed in ensuring aberrant binucleate cells do not form in syncytial cells and the different modes of nuclear the absence of budding. The G2-delay is division bring different potential advantages to determined by the ratio of active ScSwe1p to the cells (Nygaard et al., 1960; Clutterbuck, 1970; phosphatase ScMih1p (Cdc25 homologue), Gladfelter et al., 2006). In synchronous division, which can reverse phosphorylation of the CDK nuclei in distant regions of the cell can be (Ciliberto et al., 2003). If proper morphogenesis coordinated and a cell can globally respond to a is not recovered after several hours, ScMih1p signal or stimulus with a limited spatial activity leads to adaptation and the unbudded distribution. In asynchronous division, the cell cells undergo mitosis and become binucleate can restrict mitosis to particular nuclei providing (Sia et al., 1996). During unperturbed cell a more local and spatially controlled response to growth, ScSwe1p is targeted for ubiquitin a signal. mediated degradation in the proteasome by phosphorylation (Sia et al., 1998). ScSwe1p is Asynchronous or local control of mitosis, which sequentially phosphorylated at multiple sites by is observed in several filamentous fungi inclu- at least three protein kinases (Sakchaisri et al., ding Neurospora crassa and Ashbya gossypii, 2004; Lee et al., 2005). If proper morphogenesis may enable “cells” or mycelia to target both is underway, ScCla4p (PAK) localizes to the growth and nuclear division into nutrient rich septin cortex at the neck and phosphorylates regions while arresting these processes in areas ScSwe1p during S-Phase, which is only found at of the mycelium lacking sufficient resources low levels during this stage (Mitchell and (Minke et al., 1999; Freitag et al., 2004; Sprague, 2001; Sakchaisri et al., 2004). In G2, Gladfelter et al., 2006). Additionally, asyn- ScSwe1p becomes abundant and potently chronous control of mitosis may allow mycelia to inhibits Clb-ScCdc28p. Before the onset of M- link morphogenesis programs, such as bran- phase, a raise in levels of mitotic Clb leads to ching patterns and septal positioning, to nuclear Clb-CDK dependent phosphorylation of division. Thus external triggers such as nutrients ScSwe1p (Sia et al., 1998; McMillan et al., 2002; and/or internal signals involving shape of the Asano et al., 2005). This promotes interaction of

CHAPTER I 9 ScSwe1p with the Polo-like kinase ScCdc5p, laevis) and by the stress response pathway (S. phosphorylation and subsequent degradation of pombe) (Rhind et al., 1997; Rhind and Russell, ScSwe1p (Bartholomew et al., 2001; Park et al., 1998; Yamada et al., 2004; Petersen and Hagan, 2003; Sakchaisri et al., 2004; Asano et al., 2005). The homologues of all morphogenesis 2005). In addition, data from frog egg extracts checkpoint factors in S. cerevisiae are present raise the possibility of a positive feedback loop, with varying degrees of homology in the genome where Clb-CDK could phosphorylate and of the multinucleated, filamentous fungus A. activate ScMih1p and by that further antagonize gossypii, which never reproduces by budding ScSwe1p activity (Izumi et al., 1992; Ciliberto et and rather is exclusively found in a filamentous, al., 2003). Efficient translocation of ScCdc5p to multinucleated hyphal form (Dietrich et al., 2004). the neck and tethering of ScSwe1p to the septin Given the absence of budding yet the filaments both require interaction with ScHsl1p conservation of all factors involved in the and ScHsl7p, which are recruited by the septins morphogenesis checkpoint, we speculated that (Barral et al., 1999; Shulewitz et al., 1999; this cell cycle module may have co-opted for Longtine et al., 2000; McMillan et al., 2002; control of nuclear density in A. gossypii and Sakchaisri et al., 2004; Asano et al., 2005). sought to identify triggers that would lead to a Thus, in S. cerevisiae, the septin scaffold func- delay in nuclear division. We show here that tions to coordinate bud morphogenesis and nuc- AgSwe1p regulates nuclear progression in lear progression by regulating the abundance of response to external nutrient status through ScSwe1p. Additionally, defects in the actin inhibitory phosphorylation of the CDK. In cytoskeleton are thought to trigger a second addition, the septin proteins may use this module signaling pathway, operating through a MAPK to link morphogenesis to nuclear cycle in this (Mpk1), which leads to inhibition of ScMih1p filamentous fungus by directing where mitosis (Harrison et al., 2001). takes place in the mycelium. We propose that this dual role for Swe1p may be used to facilitate Interestingly, similar cell cycle modules in other a spatially controlled reaction to limited nutrient model organisms are used by the DNA availability in the natural world. damage/replication checkpoint (S. pombe, X.

CHAPTER I 10 Cla4 P

Septins Hsl1/7 P Cdc5 Swe1p kinase P Y19 - P Checkpoint Checkpoint CDK Clb CDK Clb off triggered P? Mih1p Complex active Phosphatase Complex inactive Enter mitosis Arrest in G2

MAPK

[Swe1] mitotic delay ~ / [Mih1]

Figure I.1. Morphogenesis checkpoint in S. cerevisiae. The G2-delay is determined by the ratio of active ScSwe1p to the phosphatase ScMih1p. This ratio is regulated at multiple steps to link proper control of mitotic entry with morphogenetic events. See Chapter I, Introduction for detailed information. Results

Phosphorylation of Cdc28-Y18 is a candidate nously growing mycelia or mycelia that had been for regulation of the nuclear cycle artificially synchronized (approximately 70% Phosphorylation of a conserved tyrosine residue synchrony) in G2/M with nocodazole and of the CDK is a widespread way to delay the released for progression through mitosis (Figure cell- or nuclear cycle in response to a variety of I.3B). Hence, it can be assumed that CDK phos- intra- or extracellular signals, which differ phorylation only plays a marginal role during between different organisms (Rhind et al., 1997; normal growth conditions. Rhind and Russell, 1998; Yamada et al., 2004; Petersen and Hagan, 2005; Nakashima et al., AgCdc28p is phosphorylated on tyrosine 18 unpublished). Alignment of the respective se- when hyphae are starved for nutrients quence among a selection of eukaryotes re- Given the common role of CDK phosphorylation vealed Tyr18 to be a likely candidate of regula- in different checkpoint responses across eu- tory phosphorylation of the A. gossypii CDK, karyotes, we evaluated if AgCdc28p was phos- AgCdc28p (Figure I.2A). Effectively delaying the phorylated when mycelia were exposed to nuclear cycle via inhibitory phosphorylation of potential checkpoint triggers and environmental AgCdc28p would only possible, if AgCdc28p stresses including Hydroxyurea (to impair DNA represented the key regulator of the nuclear replication), nocodazole (to impede spindle as- cycle. To test the essential function of the A. sembly), starvation, osmotic shock, and high gossypii CDK, Agcdc28Δ deletion mutants were temperature (42 °C). Latrunculin A could not be created. Heterokaryotic mycelia were able to used at the required concentration for large vol- grow at normal rates under selective conditions, ume cultures and therefore was not assayed. No indicating that transformed nuclei were still able or limited phosphorylation was detected using to divide. This was in contrast to homokaryotic the anti-phosphoTyr15-cdc2 antibody on whole mycelium grown from spores: growth stopped at cell extracts from cultures treated with Hydroxy- the stage of bipolar germlings, with rare attempts urea or nocodazole (Figure I.3C). High tem- of branch formation. Hoechst staining revealed perature stress and osmotic shock resulted in up to 4 nuclei per germling (Figure I.2B top moderate phosphorylation on a protein of the right), dominated by nuclear debris which may predicted size of AgCdc28p. Surprisingly, star- be the result of nuclear breakdown (Fig I.2B vation that was induced by high-density growth large picture). Although up to two mitoses could (evaluation described in materials and methods) still occur in the deletion mutants, AgCdc28p resulted in a level of phosphorylation that was must play an essential role already early in the markedly stronger than in any other condition development of mycelium. tested (Figure I.3C). This phosphorylated form of AgCdc28p was visible both in whole cell extracts Minimal AgCdc28-Y18 phosphorylation under from high-density cultures and in the nuclei of ideal growth conditions starving intact mycelia visualized by immuno-

We asked which conditions may cause A. fluorescence (Figure I.4A). The high-density/- gossypii to phosphorylate its CDK and potentially nutrient deprivation-induced phosphorylation was delay the nuclear cycle by using a phospho- reversed, along with the phenotype associated specific anti-cdc2 antibody on mycelia and total with starvation (prominent vacuoles) but proteins. Even under ideal growth conditions, relatively slowly upon feeding the mycelia with phosphorylation of the CDK may be an integral fresh media (Figure I.4B). The phosphorylation part of nuclear cycle regulation, for instance to was reduced after 3 hours in fresh media and at maintain asynchrony of mitoses. To test this, the minimal levels found in low-density cultures phosphorylated CDK was assayed for in mycelia after 5 hours. The response was independent of by immunofluorescence to see if a subset of the type of nutrient that was restricted and was nuclei may be enriched for modified AgCdc28p observed when either carbon (AFM with 0.1% (Figure I.3A). No phosphorylated protein was glucose), nitrogen (ASD-Asn) or amino acids detected by this method but potentially this could (ASD with one quarter amino acid concentration) be due to technical problems resolving the pro- was the limiting resource even when the cultures tein with this antibody by immunofluorescence. were grown to low density (Figure I.4C). Thus, whole cell extracts from mycelia were also generated to evaluate if the antibody could To confirm that the phosphorylation observed recognize the A. gossypii CDK by western blot. was as predicted on the tyrosine 18 residue of Only minimal phosphorylation on AgCdc28p AgCdc28p, this residue was mutated to (compared to inducing conditions in later ex- phenylalanine which cannot be phosphorylated. periments) was apparent in either asynchro- The phosphorylated AgCdc28p found in high-

CHAPTER I 11 density cultures of the reference strain is not The nuclear cycle is delayed in specific present in lysates generated from Agcdc28Y18F stages of division during starvation mycelia grown to similar high-density (Figure If phosphorylation of the CDK was inhibitory, as I.4D). This indicates that the anti-phospho-cdc2 would be predicted, then we would expect a antibody is recognizing the conserved tyrosine delay or inhibition of the nuclear cycle that leads phosphorylation on the A. gossypii CDK. to a change in nuclear density when hyphae are limited for nutrients. In fact the nuclear to This phosphorylated AgCdc28p observed in cytoplasmic ratio was significantly different densely grown cultures could be formed in between high and low density grown cultures. response to nutrient deprivation or could be a Nuclei from mycelia grown to low density which density-dependent reaction to a quorum-sensing presumably were not yet limited for nutrients molecule that accumulates at high density. were an average of 4.6 μm apart whereas in Several approaches were taken to attempt to nutrient limited conditions this distance expanded distinguish between these possibilities. First, it to 10.2 μm (N>500 nuclei) (Figure I.5A). This can was examined whether cultures grown on plates only be explained by polarized hyphal growth could release factors that would inhibit growth of continuing (presumably until internal energy new cultures on the same plates. For that, AFM supplies such as lipid droplets are depleted) plates were inoculated with A. gossypii wt and while the nuclear cycle is delayed or blocked grown for ten days. The mycelium was scraped under starvation. Agcdc28Y18F mycelia which off, leaving behind a mixture of spores and small were resistant to CDK phosphorylation showed pieces of mycelium. Half of the plates were an intermediate nuclear density in low nutrients soaked with 5 ml dH2O to replace the liquid lost of 7.9 μm (N>300 nuclei) suggesting that some by evaporation. To replace the consumed but not all of the delay in nuclear division was nutrients, 5 ml of 6xAFM were added to the other due tyrosine phosphorylation based inhibition of half of the plates. If the plates contained quorum AgCdc28p (Figure I.5B). from the previous cultures, no growth should be observed on any of the plates. However, two If such a delay was stochastic such that limited days later, dense growth was observed on the nutrients can block nuclei in any stage of the plates treated with 6xAFM, but not on the nuclear cycle then high density cultures would be watered plates. This implies that growth on used expected to have similar proportions of nuclei in plates is not inhibited by quorum factors, but by each nuclear cycle stage as found in low-density nutrient deprivation. cultures. If, however, the delay was regulated such that it occurs in a specific window of time, In the next approach, cultures were treated with the proportions of nuclei in each stage of nuclear rapamycin which likely inhibits the kinase division should vary between the different growth AgTor1/2p, which has a conserved role in conditions. Under high-density growth conditions, nutrient sensing in a variety of eukaryotes. more nuclei accumulated in hyphae with Inhibition of Tor kinase mimics starvation in duplicated SPBs (41%) and metaphase spindles many cells and in yeast simulates nitrogen (16%) compared to low density cultures (28% starvation (Martin and Hall, 2005). In A. gossypii duplicated SPBs, 4% metaphase, N>400 nuclei). mycelium grown at low density in rich nutrient Thus the deprivation of nutrients leads to conditions, AgCdc28Y18 phosphorylation clearly accumulation of nuclei in specific stages of accumulated with time in the presence of division, both just prior to and during metaphase rapamycin (Figure I.4E). Thus, the appearance (Figure I.5C). of the phosphorylated form does not depend upon high density here but does appear during Homologues of the yeast morphogenesis this mock starvation. Furthermore, when mycelia checkpoint components in A. gossypii grown at low density are transferred to MOPS/- In S. cerevisiae, inhibitory phosphorylation of KCl buffer (osmotically stable but without nutria- ScCdc28p at Tyr19 by the kinase ScSwe1p is ents) to induce rapid low-density starvation, CDK the principle response to a delay in bud forma- phosphorylation appears within 30 minutes and tion (absence of proper septin collar leads to accumulates to levels comparable to high- stabilization of ScSwe1p) or perturbation of the density starvation by 105 minutes (Figure I.4E). actin cytoskeleton. Despite dramatic morphoge- Thus, AgCdc28Y18 phosphorylation appears in netic differences between A. gossypii and budd- response to nutrient deprivation rather than other ing yeast, homologues of the entire S. cerevisiae possible signals that may accumulate at high- morphogenesis checkpoint are present in A. density growth. gossypii. Whereas the CDK, the polo kinase Cdc5p and the septins are highly conserved between the two organisms, considerable diversity was observed between the homologues

CHAPTER I 12 of the Swe1 regulators Hsl1p and Hsl7p and the Only minimal role of morphogenesis check- phosphatase Mih1p (Table 1). Given that point homologues in regulating nuclear AgCdc28p is strongly phosphorylated in starving density under non-starving conditions mycelium which results in a nuclear cycle delay, We wanted to know whether the changes in we asked whether the components of this cell AgCdc28Y18 phosphorylation caused by some cycle control module have evolved in A. gossypii of the mutants were also reflected in the average to link CDK activity to availability of nutrients. distances between nuclei and the lengths of nuclear cycle stages. Single and combination AgSwe1p is responsible for AgCdc28Y18 mutants were evaluated for growth and nuclear phosphorylation density. Under normal growth conditions in rich We generated deletion mutants of the kinase media all mutant mycelia, with the exception of AgSwe1p, its putative inhibitors AgHsl1p, septin mutants, grew with wild-type like radial AgHsl7p, the septins, and the phosphatase growth rates (Figure I.6A). Nuclear density was AgMih1p to investigate their role in AgCdc28Y18 comparable to the reference (4.6 μm between phosphorylation. As in Agcdc28Y18F mutants, nuclei) in Aghsl1Δ (3.8 μm), Agswe1Δ (3.8 μm), Agswe1Δ mutants failed to accumulate phos- Agmih1Δ (3.8 μm) and Agcdc12Δ (4.7 μm) phorylated AgCdc28p in high-density conditions mutants (N> 300 nuclei scored for each strain, in contrast to reference mycelia (Figure I.7A). (Figure I.6B). The nuclear cycle phase propor- Additionally, low-density grown Agswe1Δ myce- tions were similar to the reference for the differ- lia that were incubated with rapamycin also did ent mutant strains (Figure I.6C, nuclear cycle not have detectable AgCdc28p phosphorylation stage scoring based on spindle appearance by showing that both, the rapamycin and starvation anti-tubulin immunofluorescence) however, there induced modification involved AgSwe1p (Figure was a moderate increase in the percentage of I.7A). Agmih1Δ mutants had comparable levels metaphase and anaphase nuclei in Aghsl1Δ of phosphorylation to reference mycelia (Figure (12% vs 7% in wt) and Agcdc12Δ (14%) mutants I.7A) suggesting that under these conditions suggesting there may be some delay late in the AgMih1p may be down regulated in wild type. nuclear division cycle due to the absence of Growing Agcdc12Δ and Aghsl1Δ mutants to high these proteins. density was only partially possible due to the growth defects of these mutants. As a result, Based on homologues in other systems, only moderate phosphorylation was observed. Aghsl1Δmih1Δ double mutants or double deletion Interestingly, when AgMih1p was deleted in strains of AgMIH1 and any of the crucial septins addition to Agcdc12Δ or Aghsl1Δ, the CDK would be predicted to have a synthetic inter- phosphorylation was markedly increased action due to the release of inhibition of although these double mutants were afflicted by AgSwe1p in the absence of the phosphatase, the same culturing problems as the single mu- AgMih1p, which likely opposes AgSwe1p. In tants (Figure I.7B). This implies that AgMih1p yeast the analogous mutations lead to inviable may at least be one phosphatase of the CDK cells which are blocked at the G2-M transition that keeps AgSwe1p dependent phosphorylation (McMillan et al., 1999a). Amazingly, there was in check. This would entail reduced reversibility no additive effect in terms of growth in of AgCdc28p phosphorylation in Agmih1Δ mu- Aghsl1Δmih1Δ, Agcdc3Δmih1Δ, Agcdc10Δmih1Δ tants. Experiments were undertaken to see or Agcdc12Δmih1Δ double mutant strains in A. whether Agmih1Δ single mutants or combination gossypii. Even analyzing nuclear density or mutants with Aghsl1Δ and Aghsl7Δ displayed nuclear division cycle stages did not reveal any impaired capability to recover after starvation, synthetic effect in Aghsl1Δmih1Δ or Agcdc12Δ− upon feeding the mycelia with fresh full medium. mih1Δ (Figure I.6A-C). These data combined No conclusive results were obtained because it suggest that despite the conservation of this cell was not possible to grow all the strains to the cycle control module in the genome there is only same density and therefore they were starved to a minimal “global” role for the septins/AgHsl1p/- a different degree when the recovery experiment AgSwe1p, and AgMih1p in A. gossypii for regu- was started (data not shown). In none of the lating the frequency of nuclear division under cases, however, did the mutant strains display standard laboratory (low-density / rich nutrient) reduced recovery capacity compared to the ref- growth conditions. erence strain. This indicates the existence of additional, AgMih1p independent mechanisms Starvation induced nuclear cycle delay by which the CDK phosphorylation can be depends on AgSwe1p reverted. Strong AgCdc28-Y18 phosphorylation was only observed under starving conditions, except in Agcdc18Y18F and Agswe1Δ mutants. Thus, even if components of this cell cycle control

CHAPTER I 13 module have no clear effect on the nuclear cycle AgCdc28p was readily detected in low-density under standard laboratory conditions, they may conditions when AgSwe1p was overexpressed still be involved in regulation of the nuclear cycle from the ScHIS3 promoter (lanes 2 and 4 delay we had observed under starving compared to 6, (Figure I.8B). conditions. To investigate this possibility, we evaluated average distances of nuclei in deletion To determine if this Y18 phosphorylation of mutants grown to high density (starvation). AgCdc28p could alter nuclear cycle progression Agcdc12Δ (13.3 μm), Aghsl1Δmih1Δ (14.7 μm) even in low-density cultures, nuclear density was and Agcdc12Δmih1Δ (15.5 μm) mutants all had evaluated in these hyphae overexpressing in average higher nuclear densities than the AgSwe1p. The overexpressed AgSwe1p and reference (10.2 μm) (Figure I.8C). One caveat to presumably the subsequent AgCdc28p phos- these experiments however is that the slower phorylation led to a 50% decrease in nuclear growth rate of septin mutants necessitates a density with an average distance between nuclei longer period of overall growth to achieve high of 8.4 μm compared to 4.6 μm in the reference at density and thus the growth time is not identical low density (Figure I.7C). Additionally, the between septin mutant strains and other strains. overexpressed AgSwe1p led to a dramatic delay Agswe1Δ mycelia grown to high density failed to in the nuclear cycle leading to a population in respond as strongly to these conditions as the which almost 60% of nuclei had duplicated SPBs reference (10.2 μm), but still showed some compared to only 28% in reference low density increase in average nuclear distance (8.0 μm) cultures further suggesting that AgSwe1p- compared to non-starving conditions (3.8 μm). induced AgCdc28p phosphorylation acts prior to This was comparable to the 7.9 μm measured in metaphase (Figure I.8A). starving Agcdc28Y18F hyphae (Figure I.7C). Agswe1Δ mutant mycelia had fewer nuclei with Mitosis are concentrated at sites of branch duplicated SPBs than reference hyphae at high formation density but retained a similar proportion of So far, we have not succeeded to clearly show mitotic nuclei (Figure I.8A). These results involvement of the septins or AgHsl1p and suggest that AgSwe1p induced AgCdc28Y18 AgHsl7p in regulating AgSwe1p activity, mainly phosphorylation is responsible for a delay in due to technical difficulties. Nevertheless, they nuclear division in G2 and that some other are the key regulators of ScSwe1p in budding factors are responsible for the increased yeast. Are the A. gossypii homologues of these proportion of metaphase nuclei in high-density proteins involved in a way of AgSwe1p regulation growth. which is far more subtle than starvation response? One way how the septins could To determine if Swe1p activity was sufficient for regulate AgSwe1p is by locally inactivating this AgCdc28p phosphorylation and altered nuclear kinase to provide an increase in the rate of division even in the absence of high-density mitosis in regions of growth. To see whether the starvation, AgSwe1p was overexpressed in septins are in the right place for such a mycelium grown in low-density conditions. For regulation, we localized septin proteins in A. this experiment the AgSwe1p promoter was gossypii using a GFP-tagged septin, AgSep7p- replaced with the S. cerevisiae HIS3 promoter GFP, and by immunofluorescence using an which leads to high, constitutive expression of antibody generated against ScCdc11p. Detailed proteins in A. gossypii (Dominic Hœpfner, information on septin localization is found in personal communication). Protein levels were chapter II (Figure II.2). Anticipatory, septins did assayed by western blot against a 6HA-epitope actually localize to regions of growth: bar like tag fused to the C-terminus of AgSwe1p to rings were found near growing tips and close to confirm that the HIS3 promoter leads to sites of branch formation, asymmetric structures overexpression of the AgSwe1p protein (Figure were found at the bases of branches. In order to I.8B). Additionally, AgSWE1 expression under its have a mitosis promoting effect, the septins own promoter was evaluated for regulation and would have to bring together AgSwe1p and its to confirm that the HA fusion protein was inhibitors in these regions. We attempted to functional during high and low-density growth. evaluate this by localizing AgSwe1p and one of Under control of the native promoter, AgSwe1p- its possible inhibitors, AgHsl7p. AgSwe1p was 6HA was barely detected in low-density cultures not visible when tagged either with GFP or and appeared to migrate slower compared to in epitopes in normally growing mycelia but high-density growth, where it was more AgHsl7p was visible in A. gossypii hyphae. abundant and the bulk of the protein appeared in AgHsl7-GFP expressed from the endogenous a faster migrating form that also predominated in promoter was concentrated in discontinuous the hyphae with AgSwe1p overexpressed. rings reminiscent of septin rings (Figure I.9A-D). Unlike in reference mycelia, phosphorylated Co-localization by immunostaining against GFP

CHAPTER I 14 and Cdc11p showed that in fact AgHsl7p co- promote mitosis (18% of mitoses, Figure I.11). localized with some but not all septin rings and in Hyphal septin rings, formed earlier than the no case was AgHsl7p observed in the absence branching sites, are a possibility how these of a septin ring (Amy Galdfelter, personal regions are marked. We tried to directly observe communication). AgHsl7p was not observed in the influence of septin structures on the cortical rings in either Agcdc12Δ or Aghsl1Δ frequency and position of mitoses by doing time- mutants suggesting that this likely Swe1p lapse studies of a strain in which both, nuclei and regulator is concentrated in specific regions of Sep7p were tagged with GFP (AgHPH009). In the mycelium by septins (Figure I.9E, F). order to reduce background fluorescence, we were forced to grow the mycelium on synthetic To investigate whether the rate of nuclear minimal medium (ASD) during time-lapse division is increased in these regions, we acquisition, which partially triggered the star- measured the mitotic index in tips, branch points vation response and greatly reduced the number and regions between these growth areas as of mitotic events. Therefore it was not possible to illustrated in Figure I.10A, using several get any conclusive results from this experiment methods. First we observed mycelia growing on (Movie 2). In another attempt performed by Amy agar expressing GFP-labeled Histone H4 as Gladfelter, AgCdc11p and tubulin were nuclear marker (AgH4-GFP) using time-lapse visualized in fixed mycelia by immunofluor- microscopy to follow nuclear dynamics and escence. 71% of nuclei with mitotic spindles morphogenesis over multiple hours in vivo were adjacent to either a tip, main hyphal or (Movie 1). Mitoses were frequently observed branch septin ring and 52% of nuclei with near newly emerging branches at the junction duplicated SPBs were adjacent to septin rings between the new branch and the “mother” (Figure I.10D, N=230 nuclei). hypha. Example frames from the movie are shown in Figure I.10B, where mitosis occurred at If septins are required for this spatial pattern of the branching site at 171’, 183’, 201’, and 213’. mitotic progression then mycelia lacking septins Mitoses are labeled with a box just prior to would be predicted to have a random spatial division and new daughter nuclei are then distribution of mitoses and potentially an altered outlined with circles. In 84 total mitoses captured frequency of mitoses. Amy Gladfelter observed by time-lapse, 10 (12%) were at hyphal tips that in mutant mycelia lacking the septin (11% of hyphal length), 43 (51%) were at current AgCdc12p both mitotic nuclei and nuclei with or future branching sites (30% of total hyphal duplicated SPBs were no longer commonly length) and 31 (37%) were in the middle of observed at branch points as seen in the hyphae (in interregion not near tip or branch reference strain and instead appeared to be sites, 59% of total hyphal length). Additionally, to randomly distributed in hyphae. In reference assay mitotic frequency and position using an mycelia grown in liquid, 45% of nuclei were near alternative method carried out by Amy Glad- a branch whereas in Agcdc12Δ only 13% were at felter, reference mycelia were grown in liquid branches (N>200 nuclei). In contrast, similar medium, then processed for anti-tubulin immu- proportions of dividing nuclei in Agcdc12Δ nofluorescence to visualize mitotic spindles. In mutants (32%) were observed in growing tip this case, branchpoints were similarly enriched regions compared to the reference (30%) for mitoses with 45% of mitotic nuclei at these suggesting that the septin ring at branch points sites while tips hosted 30% of the dividing nuclei wields greater influence over the nuclear cycle and 25% were present in the interregion (Figure than the diffuse structures at the tips of hyphae. I.10C, N>200 nuclei). Notably, we observed that Thus at least certain septin rings seem to provide when mitoses occurred in the absence of spatial directions to the mitotic machinery and branching in the interregion that in many cases a potentially help to establish a sub-cellular region branch later emerged from that spot suggesting that favors nuclear progression. that certain regions of a hypha are ”marked” to

CHAPTER I 15 Table 1. Homologues of the yeast MC components in A. gossypii

Size (aa) Syntenic Key Domains* Protein A.g. S.c. Identity homolog A.g. S.c. Cdc28p 295 298 86 % yes Protein kinase Protein kinase Swe1p 727 819 52 % yes Protein kinase Protein kinase Phosphatase, Phosphatase, Mih1p 468 554 34 % yes Rhodanese like Rhodanese like Protein kinase, Protein kinase, Cdc5 708 705 73 % Yes Polo box Polo box Hsl1p 1425 1518 43 % yes Protein kinase Protein kinase Methyltransferase Methyltransferase Hsl7p 787 827 45 % yes domain domain Cdc3p 507 520 58 % yes GTP binding protein GTP binding protein Cdc10p 328 322 75 % yes GTP binding protein GTP binding protein Cdc11Ap 411 74 % yes GTP binding protein 415 GTP binding protein Cdc11Bp 408 74 % yes GTP binding protein Cdc12p 390 407 78 % yes GTP binding protein GTP binding protein Sep7p 580 551 60 % yes GTP binding protein GTP binding protein

*Domains identified using InterPro from the UniProtKB/Swiss-Prot database

CHAPTER I 16 A 10 20 30 40 ....|....|....|....|....|....|....|....| AgCdc28 MS-DLTNYKRLEKVGEGTYGVVYKAVDLR--HGQRIVALK Ashbya gossypii ScCdc28 MSGELANYKRLEKVGEGTYGVVYKALDLRPGQGQRVVALK Saccharomyces cerevisiae SpCdc2 ----MENYQKVEKIGEGTYGVVYKARHKL---SGRIVAMK Schizosaccharomyces pombe KLLA0B0979 MS-ELTNYKRLEKVGEGTYGVVYKAVDLR--HQNRVVAMK Kluyveromyces lactis CaCdc28 MV-ELSDYQRQEKVGEGTYGVVYKALDTK--HNNRVVALK Candida albicans AnnimX ----MENYQKIEKIGEGTYGVVYKARELT--HPNRIVALK Aspergillus nidulans NCU09778.1 ----MENYQKLEKIGEGTYGVVYKARDLA--NSGRIVALK Neurospora crassa BrCdk2 ----MESFQKVEKIGEGTYGVVYKAKNKV---TGETVALK Brachydanio rerio BrCdc2 ----MDDYLKIEKIGEGTYGVVYKGRNKT---TGQVVAMK Brachydanio rerio GgCdc2 ----MEDYTKIEKIGEGTYGVVYKGRHKT---TGQVVAMK Gallus gallus XlCdk2 ----MENFQKVEKIGEGTYGVVYKARNRE---TGEIVALK Xenopus laevis MmCdk2 ----MENFQKVEKIGEGTYGVVYKAKNKL---TGEVVALK Mus musculus HsCdk2 ----MENFQKVEKIGEGTYGVVYKARNKL---TGEVVALK Homo sapiens HsCdc2 ----MEDYTKIEKIGEGTYGVVYKGRHKT---TGQVVAMK Homo sapiens HsCdk3 ----MDMFQKVEKIGEGTYGVVYKAKNRE---TGQLVALK Homo sapiens

B Agcdc28∆

Figure I.2. Conserved tyrosine phosphorylation of the CDK. (A) Alignment of N-terminal sequences of cyclin dependant kinases among a selection of eukaryotes. The arrow indicates the phosphorylatable, conserved tyrosine residue (Tyr18 in AgCdc28p). (B) Hoechst staining of Agcdc28∆ mutants (AgHPH35), grown for 10 h in liquid AFM at 30 °C. A low density culture, DNA low density culture, AgCdc28-P

B Figure I.3. Minimal role of AgCdc28-Y18 le o phosphorylation under ideal growth condi- , z e e y a e e s s d t s s a a tions, but strong phosphorylation during e i d t s o a a le le a n c le le e e starvation. (A) The reference strain (∆l∆t) was e e o r r r re re ' ' t d N grown under low density conditions and stained n ' ' 5 0 u w h o 5 0 3 8 with Hoechst for DNA visualization (left) and as- l 4 4 9 1 1 + sayed for phosphorylated CDK by immunofluorescence using the anti-phospho-cdc2(Tyr15) an- loading tibody (right). Bar, 10 µm. (B) Protein extracts control from ∆l∆t mycelia grown for 11 h under low density conditions then arrested and released from nocodazole (4 h) were assayed for phosphory- C lated CDK on a Western blot, using the anti- , , phospho-cdc2 (Tyr15) antibody. A non-specific , , a a e e e e band from the ponceau red treated membrane y l y l r y r it y o it o y it y it it u u it was used for the loading control. Extracts from , s , z s z y s y g n g s a n a s n s n n x x n the yeast strain DLY5544 (cdc12-6, PGAL1- u e u d e d o e o r d r e o d o e r d r e d d d c c d d d d SWE1) served as size control (+) for this and all h h h o o o o y y ig w ig w ig w following westerns. (C) ∆l∆t mycelia were n n o N N o H H o h l h l h l + grown for 15 h at 30°C before exposure to checkpoint stimuli or environmental stresses followed by lysis and Western blotting of whole c e l l e x t r a c t s u s i n g t h e a n t i - p h o s p h o- loading control cdc2(Tyr15) antibody. When not indicated otherwise, the cultures were grown at low density before applying the various stresses. Nocodazole M M 5 0 was used at 15 µg/ml, hydroxyurea at 50 mM. 2 5 ty C C . . ty i ° ° i As loading control, a non-specific cross reacting s 0 0 s s 2 s 2 s l l n s s s C C n e 4 4 e band was used for the blot at the top, non-spe- d re t re t re a a d t a t a t N N h s s s h cific band from the ponceau red treated mem- ig o h o h o h h ig h n 4 n 3 n 1 1 h brane was used for blot at the bottom.

loading control A high density culture, DNA high density culture, AgCdc28-P

B starving 3 h recovery 5 h recovery ry ry e e v v g o o n c c i e e rv r r ta h h s 3 5 +

loading control

C low density D F F 8 8 y 1 y 1 % t y t y e i e t Y i Y t .1 y s i 8 s 8 i m it c c s s 0 n n n n 2 n 2 iu s s n c c n lc acids n e e e e d A e r d r e d d d e e G - d d d e e c c m f h f g h g M D D h e e ll g ig w ig w F S S amino i r r o A A o fu A A A ASD ¼ h + h l h l

loading loading control control

Figure I.4. Starvation induced AgCdc28-Y18 phosphorylation is reversible, independent of the type of re- E stricted nutrient and not the result of quorum sensing. (A) The reference strain (∆l∆t) was grown for 16 h under high density conditions, stained with Hoechst for DNA visualization (left) and assayed for phosphorylated CDK by immunofluorescence using the anti-phospho- ' Rapamycin cdc2(Tyr15) antibody (right). Bar, 10 µm. (B) ∆l∆t myce- low density low density05 high density low density 30' Rapamycin 1 30' MOPS/KCl105' MOPS/KCl lia were grown under high density conditions in 20 ml of AFM until prominent vacuole formation was observed (18 loading h). The mycelia were washed and resuspended in 200 ml control of fresh AFM to recover. Samples were taken after 3 h and 5 h, cells were lysed and the levels of CDK phosphorylation in whole cell extracts were detected by Western blotting using the anti-phospho-cdc2(Tyr15) antibody (left). A non-specific cross-reacting band was used as loading control. The degree of vacuolization was observed by phasecontrast microscopy (right). (C) Low density ∆l∆t mycelia were grown in full medium (AFM) for 12 h before shifting them into media limited for different resources: AFM Glc 0.1% (20 times less glucose than standard grwoth media), ASD(synthetic media without yeast extract), ASD-Asn (synthetic media lacking asparagine), and ASD with 0.25 normal amino acid concentration. Samples were taken 5 h after the media shift and assayed for CDK phosphorylation as described above. (D) ∆l∆t and Agcdc28Y18F(AgHPH36) mycelia were grown to the indicated densitites, lysed and processed by Western blot using the anti-phospho- cdc2(Tyr15) antibody. A non-specific cross-reacting band was used as loading control. (E) The reference strain was grown for 15 h under low density conditions before adding 200 nm Rapamycin or transferring the cells to MOPS/KCl (40 mM MOPS [pH 7.0], 137 mM KCl) except for the left lane where the culture was grown to high density for a positive control. CDK phosphorylation was detected by Western blotting as described above. A Reference, low density culture, DNA: 4.6 µm/nucleus Reference, high density culture, DNA: 10.2 µm/nucleus

B Agcdc28Y18F, low density culture, DNA: 4.7 µm/nucleus Agcdc28Y18F, high density culture, DNA: 7.9 µm/nucleus

C D

100% 3% 3% 4% Ana 90% 16% Meta Ana 2 SPB 80% 28% 1 SPB 70% Meta 60% 41% 50% 40% 2 SPB 30% 66% proportion of nuclei 20% 39% 1 SPB 10% 0%

low densityhigh density

Figure I.5. The nuclear cycle is delayed in specific stages of division during starvation. (A) Reference (∆l∆t) and (B) Agcdc28Y18F (AgHPH36) mycelia grown under high and low density conditions were grown to low and high densities, fixed and stained with Hoechst to visualize nuclei. Bars, 10 µm. (C) ∆l∆t mycelia were grown for 16 h at 30°C to high/low density and then fixed and processed for anti-tubulin immunofluorescence to evaluate the percentage of nuclei in different nuclear division cycle stages (Ana: Anaphase, Meta: Metaphase, 2 SPB = 2 spindle pole bodies, 1 SPB = 1 spindle pole body). (D) Example of scoring method used in C (red: nuclei stained with Hoechst, green: anti-tubulin). Bar, 5 µm. A ∆l∆t Agmih1∆ Aghsl1∆ Aghsl1∆mih1∆ Agcdc12∆ Agcdc12∆mih1∆ Agswe1∆

100 % 97 % 85 % 90 % 71 % 71 % 95 %

B ∆l∆t Agmih1∆ Agswe1∆

4.6 µm 3.8 µm 3.8 µm

Aghsl1∆ Aghsl1∆mih1∆ Agcdc12∆ Agcdc12∆mih1∆

3.8 µm 4.1 µm 4.7 µm 4.6 µm

C 100% 3% 4% 7% 7% 4% 4% 3% 8% 8% 4% 90% 4% 6% 7% 7% 80% 28% 28% 28% 27% 26% 25% 24% 70% 60% 50%

40% % nuclei 30% 66% 65% 61% 61% 61% 61% 64% 20% Ana Meta 10% 2 SPB 0% 1 SPB ∆l∆t mih1∆ hsl1∆ hsl1∆ cdc12∆ cdc12∆ swe1∆ -mih1∆ -mih1∆

Figure I.6. Minimal role of homologues of the S.c. morphogenesis checkpoint components. (A) Radial growth on solid medium of the reference strain (∆l∆t) and deletion mutants Agmih1∆ (Ag- HPH23), Aghsl1∆ (hsl1∆NAT1), Aghsl1∆mih1∆ (AgHPH28), Agcdc12∆ (AgHPH15), Agcdc12∆mih1∆ (AgHPH27), Agswe1∆ (ASG35) compared to ∆l∆t (100 %). A small piece of homokaryotic mycelium (~1 mm3) from each mutant was spotted on full medium plates and grown for 4 days. (B) Nuclei visualized in different strains. Mycelia (same strains as above) were grown overnight at 30°C in selective, liquid medium, fixed and processed for Hoechst dye staining. Numbers indicate average distance between two nuclei, which was determined by dividing variable lengths of hyphae by the number of nuclei observed in such a segment. (C) Percentage of nuclei (N>400) in different nuclear division cycle stages evaluated by Hoechst and anti-tubulin staining (Ana: anaphase; Meta: metaphase; 2 SPB: 2 spindle pole bodies; 1 SPB: 1 spindle pole body). A B y y it y it y s it s it y s s ty t ty n n i i i e n e n s ∆ s ∆ s d e d e 1 d d n n 1 n h h e e h e i e ig w ig w d w d d o o h l h l Ag cdc12 ∆ s m h g h g h g g g ∆l∆t i A i A i ∆l∆t Agswe1∆ h h h + 180' Rapamycin 180' Rapamycin Ag hsl1 ∆

loading loading control control Agmih1∆

C ∆l∆t, high density Agswe1∆, high density ∆l∆t, low density PScHIS3-AgSWE1, low density 10.2 µm/nucleus 8.0 µm/nucleus 4.6 µm/nucleus 8.4 µm/nucleus

Figure I.7. AgSwe1p is responsible for AgCdc28Y18 phosphorylation and mitotic inhibition in starving hyphae. (A) ∆l∆t (reference), Agswe1∆ (AgHPH24) and Agmih1∆ (AgHPH23) mycelia were grown to high density and whole cell extracts were assayed for CDK phosphorylation as described above (left). ∆l∆t and Agswe1∆ (AgHPH24) cells were grown 15 h to low density and then incubated with Rapamycin (200 nM). A non-specific cross-reacting band was used as loading control. (B) Agcdc12∆ (AgHPH15), Agcdc12∆-Agmih1∆ (AgHPH27), Aghsl1∆ (hsl1∆NAT1) and Aghsl1∆-Agmih1∆ (AgHPH29) cultures were grown 19 h to high and low density and assayed for CDK phosphorylation. (C) Nuclei of mycelia grown for 16 h at 30 °C visualized by Hoechst staining. Bar, 10 µm.

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g 1 1 1 1 2 1 µ e a s u e l c u n / m proportion of nuclei of proportion i n 0 e e u y A A A number a ( 3 F ( c p n w w ( b A C AgHSL7-GFP Aghsl1∆ HSL7-GFP Agcdc12∆ HSL7-GFP A D E F Phasecontrast

B GFP C Merge

Figure I.9. AgHsl7p localizes to septin-dependent cortical rings. (A-D) AgHSL7-GFP (ASG41) mycelia were grown for 12 hours at 30°C and then imaged live. (B) 90° 3D reconstruction of the discontinuous ring shown in (A). (E, F) Aghsl1∆, AgHSL7-GFP (AgHPH20) and Agcdc12∆, AgHSL7-GFP (AgHPH19) were grown for 12 hours at 30°C and then imaged live. Phasecontrast, fluorescence and merged images are ordered from left to right (A) or from top to bottom (C-F). Bars, 10 µm. A T I FB I B I B I

B 168' 171' 174' 177' 180' 183'

186' 189' 192' 195' 198' 201'

204' 207' 210' 213' 216' 219'

C D DNA tubulin 70% wt Agcdc12∆ G2 M 60% Agswe1∆ G2 Aghsl1∆ M i

e Agmih1∆ l 50% G2 c u n

40% c i t o

t 30% i m

% 20%

10% 5% 3% 6% 2% 2% 0% 4 1 1 2 2 30% 28% 22% 31% 26% 25% 59% 59% 47% 52% branch tip interregion AgCdc11p merge

Figure I.10. Mitoses are enriched at branching points. (A) Scheme (designed by Peter Philippsen) showing the different regions of a developing mycelium that were considered for determining the spatial mitotic index: tip (T), interregion (I), future branch site (FB) and branch site (B). Tip nuclei were the 2 nuclei closest to the growing tip, branch/future branch nuclei were within 5 µm of a branch site (10 µm zone) and nuclei that were not within the tip or branches were scored as in the inter-region. (B) Myelia expressing GFP-labeled Histone H4 (AgH4-GFP) as nuclear marker were grown at 25°C on diluted medium containing agar and images were collected at 3 min intervals. Hyphal outlines are in red and nuclei are in green, mitoses are labeled with a box just prior to division and new daughter nuclei are outlined with circles. (C) Percentage of mitoses found at sites of branch formation, at tips and in the inter-region of hyphae evaluated by Amy Gladfelter by scoring Hoechst and anti-tubulin stainings (N>200 nuclei). (D) Reference strain mycelia (∆l∆t) were grown overnight for 16 hours at 30°C and processed for anti-tubulin and anti-Cdc11p immunofluorescence (Amy Gladfelter). In overlay, DNA is blue, tubulin is red and septins are green. Bars, 10 µm. 252' M 282' M 312' 342'

255' 285' 315' 345' M

258' M 288' 318' 348'

261' 291' 321' M 351' M

264' 294' M 324' M 354' M

267' M 297' 327' 357' M

270' 300' M 330' M 360'

273' M 303' 333' 363'

276' 306' 336' 366'

279' M 309' 339' M 369' M

Figure I.11. Localization of mitoses at future branch sites. Mitoses in AgH4-GFP mycelium were monitored by in vivo time-lapse microscopy (Movie 1) as described for Figure I.10. Panels that contain a mitotic event are highlighted by a blue "M". Bars, 10 µm. Rich nutrients laboratory conditions Low nutrients laboratory conditions A D 1 SPB 2 SPB mitosis AgCdc28Y18-P septin structure

branch formation, wild type

Natural environment E

mature branch, wild type

+N C +C +C

+N +N

+C +C

branch formation, Agcdc12∆ feast or famine hypothesis

Figure I.12. Model of spatial control of mitosis by septins and nutrient availability. (A) Wild-type mycelia grown in the laboratory have mitoses enriched at branch sites near assembled septin rings. (B) As branch sites mature, new septin rings acquire mitosis promoting capacity, leading to mitotic events in interregions. (C) Mitoses are no longer localized preferentially to branch sites in mycelia lacking septins. (D) In low nutrient conditions, nuclei are arrested or delayed with duplicated SPBs and high CDK-Tyr18 phosphorylation levels. (E) Hypothesis for how mitoses may be spatially controlled in response to heterogeneous nutrient supplies in the natural world, where a single mycelium may simultaneously experience "feast and famine" conditions. Local pools of nutrients may trigger clustering of receptors/signaling factors for sensing nutrient status which promote local mitosis through the down- regulation of AgSwe1p, enabling the cell to exploit nutrients without duplicating nuclei distant to nutrient source. Discussion

In this work, we have sought to identify response level, however ScSwe1p can act mechanisms for how nuclear density may be independently of these pathways in filamentation regulated in multinucleated hyphal cells. We (Ahn et al., 1999). How ScSwe1p senses nutrient have shown that mitosis is limited in response to status in budding yeast has not been published nutrient deprivation, and that this is achieved so far, but there is strong data suggesting that through inhibitory AgCdc28Y18 phosphorylation, the inactivating kinase of ScSwe1p, ScCdc5p, is promoted by the protein kinase AgSwe1p. We under control of TORC1 (Nakashima et al., have shown further that mitoses are concen- unpublished). In fission yeast, nutrient limitation trated in the vicinity of branching sites, where signals through a MAP kinase called Spc1 and they may be promoted by the septins. Here we triggers a SpWee1-mediated G2 delay potentially discuss these data and propose a hypothetical through the regulation of SpNim1 (Shiozaki and model in which local regulation of AgSwe1p Russell, 1995; Belenguer et al., 1997). A. activity may enable the multinucleated mycelium gossypii is constitutively filamentous and unlike to react to a spatially limited pool of nutrients and in S. cerevisiae strains, filamentous morphology restrict mitoses to the area of the nutrient source changes are not observed under nutrient depriv- (Figure I.12). ation. Nevertheless, AgSwe1p activity makes dramatic contributions to the nuclear division AgCdc28p is the only cyclin dependent kinase in cycle kinetics in starving A. gossypii mycelium. It A. gossypii. Although the CDK is essential for was remarkable that inhibition of the Tor1/2p by the nuclear division cycle, heterokaryotic dele- rapamycin could activate Swe1p similarly to tion mutants are able to grow normally under nutrient deprivation suggesting that Swe1p selective pressure, which is only possible if the activity in response to starvation may be resistance marker used for the deletion is spread regulated downstream of this master-regulator of over the entire mycelium. This indicates that cell growth. This Tor1/2p path and/or a MAPK/- transformed nuclei are able to divide in hetero- RAS/cAMP path could communicate nutrient karyotic mycelium, despite the lack of the gene status and influence AgSwe1p in a variety of encoding the crucial kinase. This can either be ways either by influencing its stability, its explained by AgCdc28p being able to travel from localization, its transcription, its intrinsic kinase wild-type nuclei into transformed nuclei, or by activity, or affinity for AgCdc28p. Future work will cytoplasmic localization being sufficient to per- be aimed at understanding how this conserved form the essential functions. kinase senses and responds to nutrient fluctuations. We observed that AgCdc28p is phosphorylated on the Y18 residue in high-density growth or We have studied the distribution of mitoses in conditions that mimic starvation and this is fixed and living mycelium. Both methods correlated with an accumulation of nuclei with revealed a preference of mitosis to occur at duplicated SPBs and metaphase spindles and a branching sites. This could simply be due to decreased nuclear density (Figure I.12D). cytoplasmic increases that trigger a cell-size cue Overexpression of AgSwe1p is sufficient to that promotes mitosis. However, this would not induce some changes to the nuclear division explain why nuclei do not have a similarly cycle, the delay in G2, even without nutrient pronounced preference for division near growing deprivation. tips, nor would it explain why branching sites already have a mitosis promoting effect even How might AgSwe1p activity be enhanced by before the branch emerges. It has been shown nutrient limitation? In budding yeast cells, previously (Knechtle et al., 2003) that polari- ScSwe1p participates in the filamentous differ- zation sites, such as branching points, do not entiation response to nutrient limitation through occur randomly but at preformed landmarks in A. inhibiting CDK/Clb2 complexes which extends gossypii. It could be the same landmarks that G2 and promotes hyperpolarized bud growth locally promote mitosis to provide nuclei which (Edgington et al., 1999; La Valle and Wittenberg, will be required when the branch emerges. 2001). It is not clear how the status of environ- Throughout eukaryotes, septins are among the mental conditions is transmitted to ScSwe1p and first proteins that localize to polarization sites its regulators that are partially responsible for where they serve as a scaffold for a vast number sustaining this differentiation program. Both the of proteins. Recent studies have shown that A. STE MAPK and the RAS/cAMP pathways have gossypii septins are no exception (Andreas known “anchors” at the plasma membrane Kaufmann, personal communication). Together sensor level and the nuclear transcriptional with the localization pattern of the septins in A.

CHAPTER I 17 gossypii (see Chapter II), this makes them good would not be able to promote mitoses at candidates to establish landmarks for branch branches, both of which result in random division formation. In agreement with this view, septin phenotypes. Furthermore, the localization of deletion mutants, which are missing organized AgHsl7p, which functions in S. cerevisae as an septin rings, have a random spatial distribution adaptor that helps bring ScSwe1p to the septins of mitoses. However, septin rings are not only where it is targeted for degradation, is suggestive found near existent and future branching sites that the links in this regulatory system may be but also at growing tips. Why do these structures preserved in A. gossypii. Thus, in response to not promote mitosis? Septin proteins have dif- some cell intrinsic patterning signals that direct ferent appearing organizations depending upon branching in rich nutrient conditions, there is their location in hyphae and potentially these evidence that the septins may direct mitosis morphologically different structures are also spatially through AgSwe1p activity. functionally distinct. Rings composed of elongated bands form at the tips, more compact There are some problems, however, in overlay- rings of short septin bars appear more distant ing the details of the yeast checkpoint system from the growing tip along the main hyphae and onto the A. gossypii spatial control system that asymmetric bar structures are featured at most functions in response to cell intrinsic cues. branchpoints. Conceivably, these different varie- AgHsl7p is not observed at all branch septin ties of septin rings could recruit different pools of sites and rather seems to be more common at proteins, attract the same proteins with different septin rings on the main hyphae although this affinities, or function to delineate different plas- could just be due to detection problems. Addi- ma or organelle membrane domains. Support for tionally, the AgHsl7p has a key residue differ- functional difference in the structures comes ences in its sequence compared to the ScHsl7p from the observation that only mitosis frequency at phenylalanine 242. When ScHSL7F242 is near branches, not at tips, is diminished in septin mutated to leucine, the same substitution ob- deletion mutants. Thus, the rings of the main served in the A. gossypii sequence (Appendix hyphae near branches and potentially the asym- 3.6, arrow), ScHsl7p no longer can interact with metric structure that decorates the bases of ScHsl1p, and Swe1p is no longer targeted for branches may be directing mitoses in this area. degradation in yeast cells (Cid et al., 2001b). Notably, this influence over the nuclear division This raises the question as to whether AgHsl1p cycle is transient and is most pronounced as the and AgHsl7p are able to form a stable complex branch tip emerges, as seen in Movie 1. As the that could promote AgSwe1p recruitment. More branch matures, while the ring persists there are notably, when mycelia are grown in high nutrient fewer mitoses in this area suggesting a decay of conditions, no phosphorylated AgCdc28Y18 is the local mitotic inducing potential with time. This detectable by immunofluorescence and only a coincides with subtle changes in the structure of faint band is seen on Western blots. This implies the asymmetric ring (greater distance between that strong Cdc28Y18 phosphorylation does not the two halves of the ring, see Chapter II, Figure occur under high nutrient conditions, leaving only II.3A) however it is unclear if this change is little room for changes in phosphorylation levels relevant for mitosis regulation. caused by spatial control of mitosis. It is amazing that this control system is based on very subtle How might the septins locally promote mitosis at changes of Cdc28Y18 phosphorylation, although branching sites? Based on the morphogenesis AgSwe1p is capable of much stronger phos- checkpoint in S. cerevisae and conservation of phorylation of its substrate, as we have seen in these factors, it is easy to hypothesize that starving mycelia. It is conceivable that such a septins direct mitosis through the recruitment system is fragile and therefore cannot be of great and local inactivation of Swe1p. Potentially in importance for the fitness of A. gossypii unbranched areas of mycelia, some nuclei are mycelium, at least not under laboratory condi- delayed in division by active AgSwe1p. This tions. This is endorsed by none of the analyzed delay then is relieved upon septin ring formation single or double deletion mutants having any at branchpoints which leads to a local depletion/- noticeable effect on nuclear density or distribu- inhibition of AgSwe1p and then nuclear division tion under rich nutrient conditions. With that, A. at such sites. The observation that deletions of gossypii is in clear contrast to budding yeast, AgSWE1, AgHSL1, AgMIH1, or AgCDC12 simi- where the double deletion of the phosphatase larly shift mitoses away from branches to ScMih1p and the ScSwe1 inhibitor ScHsl1p in random positions supports the possibility that the leads to a lethal block at the G2-M transition. balance of AgSwe1p activity controls the spatial However, even if A. gossypii does not depend on pattern of mitosis. Mycelia lacking Swe1p would spatial control of mitosis, we would expect to see no longer have the means to limit mitoses in at least some influence on the distribution and/or unbranched areas and Mycelia lacking septins density of nuclei when this control mechanism is

CHAPTER I 18 eliminated. The fact that this is not the case conditions. In many fungi, branching is induced suggests the existence of redundant, yet uni- by exposure to pockets of nutrients allowing the dentified mechanisms to regulate nuclear mycelium to forage and exploit that presumably division and distribution. If local control of mitosis limited source. A mycelium would ideally target is abolished, this could be compensated by other these resources locally, producing more nuclei to “global” ways to decrease AgSwe1p activity, support the exploring hyphae while the rest of coupled with more efficient distribution along the nuclei and mycelium remain quiescent. In a microtubuli. Another possibility of compensation syncytium, however, it is unclear how a myce- is offered by controlling where and how often lium may delineate the active and inactive branch formation occurs. Evidence for interde- regions. Our current laboratory culturing condi- pendence of branch formation and mitosis has tions of A. gossypii make it difficult to simulate already been provided in other filamentous fungi the nutrient heterogeneity such mycelia would (Müller et al., 2000; Westfall and Momany, 2002; encounter in the natural world. However, we Dynesen and Nielsen, 2003). Notably, these have attempted to integrate the data we have mechanisms can be bypassed or overstrained generated on Swe1p spatially directing mitosis in by overexpressing AgSWE1 from to ScHIS3 the presence of abundant nutrients and Swe1p promoter, which results in decreased nuclear inhibiting mitosis during starvation into a model. density even under non-starving conditions. In this model, sudden increases in the pools of AgSwe1p activity could globally be restricted on nutrients would lead to branch initiation and a a transcriptional level in addition to locally promotion of mitoses in the limited area. Inter- controlling protein inhibition/degradation. This estingly, the Swe1p-dependent response to low transcriptional control would no longer be func- nutrients was exacerbated in mycelia lacking tional when AgSwe1p is expressed from an septins. Thus, the septins which reside close to exogenous promoter. Alternatively or addition- and at the branching sites might regulate the ally, the septins may interact more directly with Swe1p-dependent response to nutrient status. nuclei at branches by capturing the ends of However, considering the problems we were astral microtubules. Such associations have facing when growing the septin mutants, this been observed by tubulin immunofluorescence data has to be handled with care. The involve- of A. gossypii mycelia (Amy Gladfelter, personal ment of the TOR complex in starvation triggered communication) and potentially such physical phosphorylation of AgCdc28Y18 makes it more connections which may generate tension on the likely that this response is spatially controlled by astrals could be a trigger for mitosis. Swe1p may clustering of receptors/signaling factors for sen- itself have other targets than AgCdc28 that are sing nutrient status on the cell surface, which relevant for nuclear cycle progression and then inactivate a pool of AgSwe1p to generate potentially Swe1p-dependent phosphorylation mitoses locally (Figure I.12E). impacts their activity and in turn progression. Or AgSwe1p may be able to directly inhibit AgCdc28p independent of Y18 phosphorylation and there is actually precedent for this alternative inhibition of the CDK in the morphogenesis checkpoint in budding yeast (McMillan et al., 1999b).

Whereas spatially controlled mitoses do not seem to be an important feature for A. gossypii grown in laboratory conditions, this may be different in its natural environment. Filamentous fungi inhabit heterogeneous environments in the natural world. There is spatial and temporal irregularity in many factors such as water, temperature, minerals, pH in addition to carbon and nitrogen sources. A. gossypii is found outside the laboratory as a tropical plant pathogen where it is transmitted by sucking insects and can infect cotton and citrus fruits leading to dry rot (Ashby and Nowell, 1926; Batra, 1973). Given the large potential size of a filamentous fungal mycelium in which many interconnected hyphae share a common cytoplasm, a single mycelium may simultaneously experience “feast and famine”

CHAPTER I 19 Chapter II

Septins – cytokinesis factors in the absence of cell division in A. gossypii

Introduction

A characteristic feature of filamentous fungi is phosphoinositide binding motif, a conserved core their multifaceted morphology. Some are able to comprising a GTP-binding domain, a septin- choose between yeast-like growth, pseudohy- unique element and a C-terminal extension in- phal growth or true hyphal growth. Others, like A. cluding a predicted coiled coil (Saraste et al., gossypii, are restricted to the hyphal form, which 1990; Field and Kellogg, 1999; Zhang et al., is most different from unicellular growth and 1999; Casamayor and Snyder, 2003; Versele et propagation. It is of high interest to study factors al., 2004). that are involved in morphogenesis of these organisms. A protein family of such morpho- Micrographs of purified filaments raised the genesis factors are the septins. They were possibility that the septins are organized in discovered in 1970 by Hartwell and colleagues in parallel to the mother-bud axis. The 10-nm a screen for ts mutants affecting cell division striations seen on electron micrographs may be (cdc mutants). The screen revealed four mutants the result of lateral interaction between the which prevented cytokinesis at restrictive tem- filaments (Frazier et al., 1998; Longtine et al., perature. The corresponding genes represent 1998; Gladfelter et al., 2001b). Mutant strains the four original septins, ScCDC3, ScCDC10, lacking factors important for septin organization ScCDC11, and ScCDC12. Despite disrupted support this view. Instead of continuous rings, cytokinesis, the cells continued budding, DNA the septins form bars oriented along the mother- synthesis, and nuclear division, which resulted in bud axis in deletion mutants of ScGIN4, ScNAP1 large multinucleated cells with multiple, elon- and ScCLA4 (reviewed in (Longtine et al., 2000; gated buds (Hartwell et al., 1970; Hartwell, Gladfelter et al., 2001b). 1971a; Hartwell et al., 1974). By analyzing electron micrographs in 1976, Byers and The septins act as a scaffold, recruiting a Goetsch discovered ~20 evenly spaced stria- plethora of proteins (reviewed by (Longtine et al., tions of 10-nm filaments around the mother-bud 1996). These protein complexes are involved in neck in wild-type but not septin-mutant cells. cytokinesis, chitin deposition, cell polarity (re- Immunofluorescence studies revealed that the viewed by (Faty et al., 2002), spore formation septin proteins colocalize into a ring at the neck (Fares et al., 1996), in the morphogenesis (Byers and Goetsch, 1976; Haarer and Pringle, checkpoint (see Chapter I), spindle alignment 1987; Kim et al., 1991; Longtine et al., 1996; checkpoint and bud site selection (Hartwell, Field and Kellogg, 1999). The localization of all 1971a; Byers and Goetsch, 1976; Fares et al., four septins is disrupted in conditional Sccdc3 1996; Kartmann and Roth, 2001; Faty et al., and Sccdc12 mutants, indicating interdepend- 2002; Dobbelaere and Barral, 2004; Douglas et ence of the septin proteins. Strong support for al., 2005; Gladfelter et al., 2005). this finding was provided by biochemical studies: The four original septins co-purified on affinity Cytokinesis is driven through two septin columns, together with a fifth septin protein, dependent, redundant processes: recruitment ScSep7p or ScShs1p (Carroll et al., 1998; and contraction of the actomyosin ring and Frazier et al., 1998). Purified septins from bud- formation of the septum by vesicle fusion with ding yeast, Drosophila, Xenopus, and mammal- the plasma membrane (Epp and Chant, 1997; Bi ian cells are able to self associate in vitro to form et al., 1998; Lippincott and Li, 1998b, 1998a; highly ordered, filamentous structures (Frazier et Vallen et al., 2000). In contrast to septin mutants, al., 1998; Kinoshita et al., 2002; Casamayor and disruption of one single pathway only leads to a Snyder, 2003; Caviston et al., 2003; Dobbelaere delay in cytokinesis, not complete failure of cell et al., 2003; Versele et al., 2004). How the sept- division. Hence, the septins are predicted to act ins interact in vitro to form heteropentamers that at the most upstream level of cytokinesis. assemble into filaments was studied in detail in S. cerevisiae (Versele et al., 2004). Based on The role of septins in cell polarity is reviewed in these and former studies, the septins are com- detail by (Faty et al., 2002). After the apical- posed of a variable N-terminus with a basic isotropic switch in budding yeast, cortical

CHAPTER II 20 components, supposedly of the exocyst and Since their discovery in S. cerevisiae, septin polarisome, are delocalized from the apical pole homologues have been found throughout the to the entire plasma membrane of the bud, but eukaryotes, with the exception of plants. The not the mother cell. The septin ring at the neck variety of different shapes that septins can serves as a cortical barrier that prevents assume within a single cell is especially apparent membrane diffusion of these factors between the in filamentous fungi (reviewed by (Gladfelter, two compartments. This asymmetric distribution 2006). The genome of C. albicans encodes is abolished in septin mutants (Barral et al., homologues to all S. cerevisiae septins 2000; Takizawa et al., 2000; Faty et al., 2002). (CaCdc3p, CaCdc10p, CaCdc11p, CaCdc12p, CaSep7p). They form a diffuse band at the base Some conditional septin mutants do not form of emerging hyphae, a bright double ring at buds at their normal axial location. Moreover, the septation sites, an extended diffuse cap at typical localization of some bud-site-selection hyphal tips and elongated filaments stretching factors in a double ring at the neck is lost or around the spherical chlamydospores (Sudbery, disturbed in these mutants. This indicates that 2001; Kaneko et al., 2004; Martin and Konopka, the septins may serve as anchoring site for such 2004b; Wightman et al., 2004; Martin et al., factors in axially budding cells (Flescher et al., 2005). Five septins are found in A. nidulans 1993; Chant et al., 1995; Cabib et al., 1998). (AnAspAp, AnAspBp, AnAspCp, AnAspDp, AnAspEp). AnAspBp forms single rings at septa- It seems that one single septin organization tion sites that eventually split into double rings. should not be sufficient to fulfill such a variety of Additionally, AnAspBp forms a ring at sites of tasks. Accordingly, the septin cortex undergoes branch emergence which broadens into a band several changes throughout the cell cycle as the branch grows (Westfall and Momany, (reviewed by (Gladfelter et al., 2001b): The first 2002). As a effect of maturation double rings visible septin structure is a distinct ring which reflect hyphal polarity by disassembling the tip appears ~15 min before bud emergence. After proximal ring in C. albicans or the more basal bud emergence, the ring broadens to assume ring in A. nidulans (Warenda and Konopka, the shape of an hourglass around the mother- 2002; Westfall and Momany, 2002). Bases for bud neck. During cytokinesis, the septin cortex the various patterns of septin organization could splits into a double ring which eventually disap- be different modifications and/or localization of pears. How can the septin cortex undergo so different septin interaction partners. dramatic changes, although some of its functions may require it to be a stable structure? FRAP The septins in both, A. nidulans and C. albicans analysis has revealed that the turnover of control aspects of filamentous cell shape. septins at the neck undergoes multiple changes CaCdc3p and CaCdc12p are essential for prolif- during the cell cycle. The predominant, func- eration in yeast or hyphal forms. Cacdc10Δ and tional conformation is characterized by a low Cacdc11Δ mutants are viable but show aberrant turnover rate (frozen state), during which the chitin localization and cannot properly maintain septins are phosphorylated. Structural changes hyphal growth direction (Warenda and Konopka, require a destabilization of the septin cortex 2002; Martin et al., 2005). Conditional mutants of (fluid state) induced by dephosphorylation prior the essential AnAspBp display diffuse chitin to bud emergence, ring splitting and cell deposition and a hyper-branching phenotype separation (Dobbelaere et al., 2003). (Westfall and Momany, 2002).

The composition of the septin cortex does not Here, we add data on the function and localiza- only vary throughout the cell cycle but also along tion of the septins in a fungus which grows the mother-bud axis. This inherent polarity of exclusively in the hyphal form. Our studies have septin filaments allows concentration of some lead to the discovery of novel structures and proteins primarily to the mother side of the neck, revealed the astonishing fact that none of the some to the center and others to the bud site analyzed septin genes in A. gossypii is essential, (DeMarini et al., 1997; Gladfelter et al., 2001b). despite significant roles in morphogenesis.

CHAPTER II 21 Results

Septins are conserved in two organisms of bar rings changed their appearance depending most diverse morphology on their position within the mycelium. They were Abscission (complete cytokinesis) is not ob- long and diffuse close to the growing tips and got served in healthy, multinucleated A. gossypii shorter and more compact the further away they hyphae, at least not until the sporulation program were from the tip (Figure II.2C). In older parts of is initiated (Brachat, PhD thesis, 2003). Does the mycelium we found double rings with various this mean this Ascomycete does not require a distances between the two rings, probably full set of septin proteins as we find them in S. representing mature septa (Figure II.2B). At low cerevisiae? Interestingly, not only were homo- magnification, these double rings and some logues to all budding yeast septins identified in compact single rings had a continuous appear- A. gossypii, one of them even revealed two ance. To see whether the bars eventually fuse to homologues, AgCDC11A (AER445C) and form continuous rings, we investigated ambigu- AgCDC11B (AFR436C), as a result of tandem ous structures at higher magnification and by duplication followed by chromosome break using 3D reconstruction. Out of 28 single rings, (Sophie Brachat, personal communication; bars were identified in 25 rings. In three cases, (Dietrich et al., 2004). Pairwise alignment with evaluation was not possible due to poor picture the S. cerevisiae homologues was used to quality. The resolution of light microscopy was identify the domains described by (Versele et al.) not high enough to resolve the structure of (2004). With the exception of Cdc3p, all mitotic double rings. In some cases they appeared to be septins and their domain composition were made from a large number of short bars, but this found to be highly conserved between the two remains uncertain. organisms (Figure II.1), despite their substan- tially different life cycles. We had the possibility to study septin structures in giant hyphae of a strain expressing a Continuous AgSep7p-GFP ring at the neck of constitutively active form of AgCdc42p created budding yeast by (Köhli, Diploma thesis, 2003). We observed To understand where the septins may be re- unusually long septin filaments at the tip and a quired in A. gossypii, we first studied their double ring like structure composed of bars localization by using the AgSep7p-GFP con- (Figure II.2D). It is not unlikely, however, that struct. The GFP fusion was obtained by in vivo septin structures are influenced by mutations of recombination in S. cerevisiae (see materials AgCdc42p (Richman et al., 1999; Cid et al., and methods). We used this opportunity to in- 2001a; Gladfelter et al., 2001a; Caviston et al., vestigate whether AgSep7p was able to interact 2003). Thus, we have to be careful when with the S. cerevisiae septin complex, despite concluding from these “macro structures” to wild- the low conservation (38 % identity) between the type septin structures. C terminal extensions (CTEs) of AgSep7p and ScSep7p. Versele et al. (2004) had shown that The bar and gap organization of hyphal rings the CTE of ScSep7p is required for proper was confirmed by immunofluorescence using an interaction with ScCdc11p. Remarkably, a bright, anti-Cdc11p antibody (Amy Gladfelter and continuous single or double ring structure at the Michael Köhli, personal communication). This neck was observed which did not look differently made us wonder whether each bar consist of all from septin formations in budding yeast (Figure mitotic septins, leaving real gaps in between, or II.2A) (Field and Kellogg, 1999; Yuzyuk and whether there are various categories of fila- Amberg, 2003). This revealed that the AgSEP7 ments, made from different septin compositions. promoter is functional in budding yeast and the In the latter case it was conceivable that the protein does properly localize and not disturb the septins actually form continuous rings which assembly of the S. cerevisiae septins, despite appear as bar-like when only one septin is the differences in the CTE. labeled (Figure II.2E). To distinguish between the two possibilities, AgSep7p-GFP and AgCdc11A/Bp Intermitted hyphal AgSep7p-GFP rings in A. were co-immunostained using anti-GFP and anti- gossypii Cdc11p antibodies. Due to high background and Surprisingly, very different structures were ob- low signal intensity we found only one ring were served when we studied the localization of both stainings were useable. In this ring we could AgSep7p-GFP in A. gossypii, using strains car- clearly observe co-localization of AgSep7p-GFP rying plasmids or integrated constructs. Most bars and AgCdc11A/Bp bars. This puts the first noticeably, ring-like structures were not con- hypothesis (Figure II.2E, left) in favor, but is not tinuous like in budding yeast, but rather made sufficient to exclude the second hypothesis. from 9 – 12 parallel bars (Figure II.2B). These

CHAPTER II 22 Variety of septin organizations to a double ring (Figure II.5A). Remarkably, the Septins in A. gossypii were not only found to single ring was fading around 100 min before the localize as single and double rings at the hyphal signal reappeared as a double ring. The best cortex, they also formed a variety of asymmetric result was obtained with a movie of GFP labeled structures at the base of branches. Depending nuclei together with AgSep7-GFP (Movie B, on the age/length of the branch, these structures Chapter I). Using minimal medium (2xASD) in- had different appearances (Figure II.3A). Already stead of full medium resulted in less background at the base of branches that were just about to at the cost of reduced growth. Nevertheless we emerge, we observed septin localizations in the could follow the basic steps of septin deposition angles of the branch with the mother hyphae but during hyphal growth: A broad septin signal not in between (Figure II.3A, first picture). It is appeared at sites of branch formation when the not clear, whether this was an imaging artifact or first bulging of a new branch was visible. When whether the two localizations were actually not the bulge took hyphal shape, the septin deposit connected in most branches. In some cases it was split in two parts, one portion staying at the looked as if the two deposits were about to join base of the branch, the other moving together each other to form a ring like organization. With with the growing tip. Eventually, a portion parted proceeding growth of the branch, a diffuse ring from the deposit at the tip to form a hyphal ring, appeared at the growing tip. In longer branches, whereas the other part remained at the tip the septin localization at the base of the branch (Figure II.5B). We tried to study these events in was usually joined by a ring composed of bars detail, by concentrating on two branching sites on top of it, with various distances between the with acceptable quality. At each time point (Δt = 2 two formations. At the base of mature branches min), we analyzed each focal plane individually we could observe double rings which looked like (5 planes covering 3 µm). The sketch shown in those seen in other old parts of mycelium, distal Figure II.6 was drawn based on these obser- from the base. Despite the different appearance vations. In contrast to long exposure, high of hyphal septin rings and the asymmetric septin magnification picture stacks, signal strength and organizations at the base of branches, both resolution in the movie were often too low to formations eventually lead to double rings of distinguish single septin bars and only a diffuse comparable appearance. glow was visible, which is represented as such in the sketch. Analysis of both, fluorescence and Another phenomenon, small rings or patches phase contrast planes revealed that fainting of with a diameter of 0.5 µm, was only seen in the structure and transition to a double ring mycelium expressing AgSEP7-GFP from plas- occurred at about the same time as septum mid and thus likely was an artifact due to local formation. This lead to a sandwich of two bright overexpression. These rings were of remarkable septin rings with the septum in between similarity with septin structures found in other (indicated as red in Figure II.6). More movies will organisms under some unnatural conditions be required in order to confirm this data. (Figure II.3B) (Kinoshita et al., 2002; Kozubowski et al., 2003). One ring to rule them all The manifoldness of septin structures – many of The commonly observed A. gossypii septin them so far not described in wild-type budding structures are summarized in Figure II.4A and yeast – made us wonder whether recruitment of put in relation to their position within the myce- other proteins is limited to only some of these lium in Figure II.4B. Most of the structures seen conformations. Amy Gladfelter has observed that with AgSep7p-GFP were confirmed by anti- AgHsl7p-GFP co-localizes with some, but not all Cdc11p immunofluorescence in fixed mycelium septin rings (personal communication). Here we (Amy Gladfelter and Michael Köhli, personal wanted to know which of the septin structures communication). are likely to be involved in the formation of actin rings. For that, we used mild fixation to stain the Chronological order of septin localization actin cytoskeleton while preserving the integrity By analyzing time-lapse movies of AgSEP7-GFP of GFP. Actin rings were limited to mature, strains we aimed at gaining further insight in the contracted septin structures and in no case was temporal organization of septin structures. Un- an actin ring observed in the absence of a septin fortunately, the AgSep7-GFP signal was rela- ring (Figure II.7A). tively week for the purpose of time-lapse analysis which – combined with the strong Does this imply that the compact septin rings are background of A. gossypii in the GFP channel – essential for actin ring formation? To answer this made it difficult to follow the formation of septin and learn more about septin functions in a fila- structures in detail. In Movie A, we were able to mentous fungus in general, AgCDC3, AgCDC10, observe the transition from a hyphal single ring and AgCDC12 were deleted in an AgSEP7-GFP

CHAPTER II 23 background. This allowed us to investigate the before mature colonies can be formed? Despite importance of single septins for the association lethality of most septin null mutants in S. into the structures we had observed. Each dele- cerevisiae, Agcdc3Δ, Agcdc10Δ and Agcdc12Δ tion resulted in delocalization of AgSep7p-GFP deletion strains form colonies on solid medium at to the cytoplasm; no distinct septin structures 30 °C and 37 °C with a moderate reduction of were visible in these mutants. (Figure II.7B) radial growth speed to roughly 70% – 80% of the reference strain (Figure II.9A). We have seen that actin rings are only found at compact septin rings. Further, septins have been We tried to get further insight in how the septins found to permanently localize to growing tips. are required for growth by studying the morphol- What is the effect of a total loss of septin ogy of small and mature mycelia in septin null structures on the actin cytoskeleton? Are they mutants. Possible effects on the nuclear cycle, important for both, actin ring formation and which could also lead to a decrease in radial polarization of actin patches? No actin rings growth, have already been discussed in Chapter were detectable in the mutants Agcdc3Δ and I. Morphological defects in small mycelia grown Agcdc10Δ (Figure II.7C). Though these strains in liquid (10 – 15 h) were already revealed when exhibited a certain resistance to the staining looking at the stainings described above. Mutant procedure, actin patch polarization was still hyphae exhibit an erratic thickness with charac- visible. Thus, while septin rings are essential for teristic bulges in the area of branch points and the formation of actin rings, the septin protein especially around the spore (Figures II.7B, 7C, pool found at growing tips does not seem to be 8A and 8B). important for the polarization of the actin cytoskeleton. Mature mycelia grown on plates (8 days) revealed invasive growth and a curly phenotype It was shown previously that even in the of hyphae, indicating reduced capability of main- absence of actin rings, malformed chitin rings, taining growth direction. Lateral branching and and aberrant septa can still be observed in apical branching occurred in septin null mutants, Agmyo1Δ mutants, permitting low sporulation but the branching pattern appeared random. levels (Helfer, Diploma thesis, 2001). In Agcdc3Δ Long stretches free of branches or only with and Agcdc10Δ mutants neither chitin ring forma- attempts of branch formation alternated with tion, nor septum formation, nor sporulation were regions of unusually dense branching. Lateral observed (Figure II.8A). Agcdc12Δ strains were branching was still ubiquitous close to growing not stained for actin or chitin, but AgSep7p-GFP tips, where in reference mycelia of the same age localization, septum formation, and sporulation only apical branching was found (Figure II.9B). were abolished in this mutant. Thus, the septin Apparently, septins in A. gossypii are involved in structures in A. gossypii are recruiting more a variety of morphogenetic processes. components than those required for actin ring formation. Unlike in Agmyo1Δ, there is no Cortical barrier? alternative way for septum formation that could In S. cerevisiae, the septins have been shown to substitute septin deletions. form a cortical barrier around the mother bud neck which prevents membrane diffusion bet- When performing microscopy of septin deletion ween mother and bud and maintains cortical mutants, we could directly observe that damage polarity. Thus, components of the exocyst and induced leakage of the cytoplasm was not re- the polarisome are restrained to the bud (Snyder, stricted to one compartment like in the reference 1989; Barral et al., 2000; Takizawa et al., 2000; strain but extended to the entire young mycelium Faty et al., 2002). We wondered whether the (data not shown). As a result, liquid cultures of reduced growth rate and erratic thickness of septin deletion strains were usually a mixture of hyphae in A. gossypii septin deletion mutants living mycelia and thin “empty shells” (Figure could be the result of absent diffusion barriers. II.8B). Similarly, cultures grown on solid medium The septins might be required to efficiently had a high content of dead material. This maintain a pool of growth components at the strongly supports the idea of septa functioning hyphal tips by preventing diffusion towards older as bulkheads to limit damage to the affected parts of the hypha (Figure II.10A). We used hyphae. Filipin to stain sterols as an indicator of membrane polarity (Wachtler et al., 2003). A. gossypii septins are not essential but Polarization of sterols at hyphal tips was clearly required for efficient growth and proper visible in reference hyphae and septin null morphogenesis mutants (Figure II.10B). Thus, the septin ring Can A. gossypii survive without such a protective close to hyphal tips in A. gossypii apparently is mechanism, or will septin mutants inevitably lyse not required to maintain sterol polarity. However,

CHAPTER II 24 it cannot be excluded that they serve as a barrier those strains, the actin cytoskeleton was difficult for certain membrane proteins. to stain in Agcdc11AΔ mycelium, resulting in cloudy images with actin patches being barely Separating the “twins” (AgCDC11A/B) visible. Amazingly, actin rings and chitin rings The fact that the genome of A. gossypii encodes were both detectable in this strain (Figure two homologues to ScCDC11 as a result of II.12A). Fitting to these results, sporulation was tandem duplication followed by chromosome not abolished entirely but only reduced to 30%. break made AgCDC11A and AgCDC11B highly Whereas deletion of AgCDC11A leads to a mild interesting candidates for functional analysis. Do septin phenotype, we did not observe any mutant both homologues occupy distinct roles, or are phenotype of Agcdc11BΔ null mutants at all. they redundant? The high degree of conserva- Morphology, actin staining, chitin staining, and tion between the two genes made it difficult to sporulation were as in reference mycelium specifically delete and verify only one ho- (Figure II.12B). Apparently, the two homologues mologue at a time. With the exception of have diverged enough to be of different CDC11A/B-I1, Primers were designed to provide importance for the development of A. gossypii best possible diversity (Figure II.11). Verification mycelium. More experiments are required to PCRs for each strain were done with primer sets determine whether AgCDC11B is expressed for both, AgCDC11A and AgCDC11B. Both ends under laboratory conditions, whether the two of each replaced ORF were verified unambigu- homologues complement each other to exercise ously, no unspecific binding to the wrong locus functions similar to the other septins, or whether was detected (Figure Appendix 1.9B). they have partially adopted novel roles, tailored In terms of morphology, Agcdc11AΔ isolates for the requirements in a filamentous fungus in exhibited high similarity to other septin null natural environment. mutants (Agcdc3Δ, Agcdc10Δ, Agcdc12Δ). As in

CHAPTER II 25 Variable Phosphoinositide- GTP-binding Septin-unique CTE N-terminus binding motif domain element

104 114 350 403 507 AgCdc3p 58% 28% 60% 36% 64% 78% ScCdc3p 110 120 360 413 520

27 36 254 307 328 AgCdc10p 75% 54% 89% 77% 85% 32% ScCdc10p 24 33 251 304 322

12 23 246 299 411 AgCdc11Ap 74% 73% 91% 84% 77% 48% ScCdc11p 12 23 247 300 415

12 23 246 299 408 AgCdc11Bp 74% 73% 91% 84% 77% 48% ScCdc11p 12 23 247 300 415

12 23 246 299 411 AgCdc11Ap 85% 75%100% 96% 98% 79% AgCdc11Bp 12 23 246 299 408

19 30 250 303 390 AgCdc12p 78% 39% 64% 76% 85% 74% ScCdc12p 24 35 263 316 407

16 27 284 344 AgSep7p 60% 20% 82% 63% 67% 38% 580 ScSep7p 13 24 288 341 551

Figure II.1. Domain composition of A. gossypii and S. cerevisiae mitotic septins. The septins are composed of a variable N-terminus with a tract of basic and hydrophobic residues constituting a phosphoinositide-binding motif, a conserved core comprising a GTP-binding domain, a septin-unique element and a C-terminal extension (CTE) including a predicted coiled coil. The domains were identified by pairwise alignment with the S. cerevisiae homologues, using the boundaries defined by Versele et al., 2004. A B ScSEP7-GFP AgSEP7-GFP

Yuzuk and Amberg, 2003

3 1 2

C 90°

D E ?

90°

Figure II.2. AgSEP7-GPF in A. gossypii and S. cerevisiae. (A) S. cerevisiae cells bearing AgSEP7-GFP on the plasmid pagHPH002 were grown in liquid YPD with selection and imaged live. The inlay shows the localization of ScSep7p-GFP as reference (Yuzuk and Amberg, 2003). (B, C) A. gossypii mycelium with integrated AgSEP7-GFP (AgHPH007) was grown for 12 h at 30°C, washed in minimal medium and subjected to in vivo fluorescence microcopy. The inlay in (B) shows a 90° 3D reconstruction of the discontinuous ring. The large picture shows how the septin rings contract the further away they are from the tip. 1: diffuse localization at the tip, 2: intermediate ring, 3: contracted ring, 4: double ring. (C) Diffuse tip localization consists of elongated septin filaments; rings distal from the tip are made from more contracted bars.(D) AgSEP7-GFP in a background of constitutively active AgCdc42+p (AgHPH016), grown as described above, 90° 3D reconstruction in the inlay. (E) Schematic representation of the two hypotheses according to which one bar represents one septin (left) or each bar is composed of all the septins (right). Bars, 10 µm. A

B AgSEP7-GFP on plasmid (AgHPH006) ScCDC10-GFP, ScBNI4 overex. anti-MmSept2, Cytochalasin D

Kozubowksi et al., 2003 Kinoshita et al., 2002

Figure II.3. Diversity of septin organizations. (A) A. gossypii mycelium with integrated AgSEP7-GFP (AgHPH007, AgHPH008) was grown for 12 h - 15 h at 30°C, washed in minimal medium and imaged live. Pictures show septin structure at branching sites, with young branches at the left and old branches at the right. Outline of the mycelium is indicated by dotted lines. (B) Mycelium bearing AgSEP7-GFP on plasmid (AgHPH006) (left) was grown as above. Small ~0.5 µm rings are only observed in plasmidic strains and have similarity with rings seen in S. cerevisiae cells with overexpressed ScBNI4 (middle) or in mammalian cells with disrupted actin cytoskeleton (right). Bars, (A) 10 µm, (B) 5 µm. A

Early Intermediate Mature

Hyphal rings

Branch points

Figure II.4. Schematic overview of septin organizations seen in A. gossypii. (A) Early, intermediate and mature structures in hyphae (top) and at branching sites (bottom). (B) Septin structures put in relation with their positions within the mycelium. Schemes are based on microscopic observations of AgSep7-GFP. A

180' 200' 220' 240' 260' 280' 300'

320' 340' 360' 380' 400' 420' 440'

350' 360' 370' 380' 390' 400' 410'

420' 430' 440' 450' 460' 470' 480'

Figure II.5. Time-lapse analysis of septins in A. gossypii. (A) AgSEP7-GFP mycelia (pagHPH02) were pregrown at 30°C in liquid AFM for 10 h and transferred to a time-lapse slide containing solid minimal medium (2xASD) at 25°C. Every 2 min, a Z-series of 7 frames was taken at 10% intensity and 150 ms exposure (Movie A). The figure was assembled from a selection of frames with a 20 min interval. The arrow indicates a single ring which splits into a double ring. (B) AgSEP7-GFP, AgH4-GFP mycelium (pagHPH09) was pregrown in selective liquid AFM for 14 h at 30°C and transferred to time-lapse slide containing solid 2xASD at 25°C. Every 2 min, a Z-series of 5 frames was taken at 10% intensity and 100 ms exposure (Movie B). The figure shows a selection of pictures with 10 min intervals, on which one can observe the division of the original septin organization at the emerging branch into three distinct parts: a structure staying at the branching site, a hyphal ring and a faint tip localization staying at the growing tip. Bars, 5 µm. Figure II.6. Scheme of development of septin structures. The drawings are based on individual Z-plane analysis of Movie B. Green: AgSep7p-GFP, red: cell wall and septum. The arrow indicates that the hyphal rings are stationary, whereas the diffuse apical localization moves together with the tip. Maturation of hyphal double rings is not illustrated, but follows a similar pattern as double ring formation at branching sites. Due to technical limitations, bars were not always resolvable in the movie, which is drawn as diffuse signal. A AgSEP7-GFP actin merge

B Agcdc3∆ SEP7-GFP Agcdc10∆ SEP7-GFP Agcdc12∆ SEP7-GFP

C ∆l∆t, actin Agcdc3∆, actin Figure II.7. Actin ring formation depends on septin rings. (A) AgSEP7-GFP (green) mycelia (AgHPH05) were grown for 13 h at 30°C, mildly fixed with parafor- maldehyde and suspected to Rho- damin-Phalloidin staining (red). (B) Agcdc3∆, AgSEP7-GFP (Ag- HPH10), Agcdc10∆, AgSEP7- GFP (AgHPH14) and Agcdc12∆, AgSEP7-GFP (AgHPH15) mycelia were grown for 12 h and monitored by in vivo fluorescence microcopy. (C) ∆t∆l (reference), Agcdc10∆, actin A g c d c 3 ∆ ( A g H P H 1 0 ) a n d Agcdc10∆ (AgHPH14) mycelia were grown for 12 h, fixed with formaldehyde and stained with Rhodamin-Phalloidin. Bars, 10 µm. A ∆l∆t, chitin Agcdc3∆, chitin

Agcdc10∆, chitin

B Agcdc3∆, intact Agcdc3∆, "empty shell"

Figure II.8. Septins are required for chitin ring and septum formation. (A) ∆t∆l (reference), Agcdc3∆ (AgHPH10) and Agcdc10∆ (AgHPH14) mycelia were grown for 12 h in liquid AFM at 30°C, fixed with ethanol and stained with Calcofluor. (B) Brightfield pictures of an intact (left) and empty (right) Agcdc3∆ mycelium (AgHPH10). Bars, 10 µm. A ∆l∆t Agcdc3∆ Agcdc10∆ Agcdc12∆ 30°C

100% 69% 70% 71% 37°C

100% 79% 73% 71% B ∆l∆t Agcdc3∆

Agcdc10∆ Agcdc12∆

Figure II.9. Morphological defects of mature septin deletion mycelia (A) Radial growth on solid medium of the reference strain (∆l∆t) and deletion mutants Agcdc3∆ (AgHPH10), Agcdc10∆ (AgHPH14) and Agcdc12∆ (AgHPH15) compared to ∆l∆t (100%) at 30°C and 37°C. A small piece of homokaryotic mycelium (~1omm3) from each mutant was spotted on full medium plates and grown for 4 days. (B) Micrographs of ∆l∆t, Agcdc3∆ (AgHPH10), Agcdc10∆ (AgHPH14) and Agcdc12∆ (AgHPH15) mycelia grown on full medium plates at 30°C for 8 days, illustrating the disordered branching pattern and wavy phenotype. Bar, 20 µm. A

active transport passive diffusion growth septin structures

wild type septin mutant

B ∆l∆t, Filipin Agcdc3∆, Filipin

Agcdc10∆, Filipin Agcdc12∆, Filipin

Figure II.10. Septins are not required for sterol polarity. (A) Scheme illustrating how the septins might be required for cortical polarity in A. gossypii. Lack of septin barriers would lead to diffusion of membrane associated material away from the tip, resulting in reduced tip extension and swollen hyphae. (B) The reference strain (∆l∆t) and deletion mutants Agcdc3∆ (AgHPH10), Agcdc10∆ (AgHPH14) and Agcdc12∆ (AgHPH15) were grown for 12 h at 30°C in full medium, stained with low concentrations of Filipin (2-4 µg/ml) and monitored after 2 min. Polarization of sterols is not affected in A. gossypii septin mutants. Bar, 10 µm. AgCDC11A CCACTGCCGCGCTGCCGCCAACGCCTGCCGGCCTTCGACGAGCAGCCGAACAGC---TA- 56 AgCDC11B GCACTCCCGGGATGCAGCATAGGCGTGCGACAGATCCTGTAATGCACGTGCGGCTATTAC 60 CDC11A-G1 CDC11B-G1 AgCDC11A -GCTGTACAAAAAGAGAA-AAAATTG-ACGCGAGCGGTTGAATTTAAGCGAACGAAATCC 113 AgCDC11B GGCCGGAATGGTGGAGAACGTTGTGGCAACCCAGCGCTT-CA-TTCA-CGAACGAAATCC 117

AgCDC11A AAGTTACACGCTATTCACATTACCGCCCCCCTACCCTTAGTGAACAGGTTAGTCCACAAG 173 AgCDC11B AAGTTGCGCGATATTCAGAGTACCGCCCCCCTACCCTTAGTGAACAGGTTAGTCCACAAG 177 CDC11A-S1 AgCDC11A GCGAGTTTACAGAGCCGAGTAGCCGCAGAGAAGCGCATAAGACGCAAACAGCATGTCCAG 233 AgCDC11B GCGAG-TAACAAAGCCGAGGAGCCTTAGAGAAGCGGATAAGACAC-AGTACAATGGCGGG 235 CDC11B-S1 AgCDC11A TATCATCGAGGCCTCAGCCGCGCTCAGGAAGAGAAAGCACCTGAAGAGGGGCATTCAGTT 293 AgCDC11B CATTATTGAAGCGTCAGTAGCACTCAGGAAGAGAAAGCATCTAAAACGGGGTATCCAGTT 295

AgCDC11A CACCCTTATGGTGGTCGGGCAGTCCGGTTCAGGAAGATCGACGTTCATCAACACGTTGTG 353 AgCDC11B CACGGTTATGGTGGTTGGACAGTCCGGGTCGGGTAGATCGACGTTCATCAACACGCTGTG 355

AgCDC11A CGGGCAGGAAGTGGTGGAGACGTCGACGACCGTGATGCTGCCCAACGACGACGCGACGCA 413 AgCDC11B TGGACAGGAGGTGGTGGAAACCTCTACAACGGTGCTGCTACCCGAAGACGACGCGGCACA 415

AgCDC11A GATTGATGTGCAGCTGCGCGAGGAAACGGTGGAGCTGGAGGACGACGAGGGCGTCAAGAT 473 AgCDC11B GATCGACGTGCAACTACGGGAGGAGACAGTGGAGCTGGAGGACGACGAGGGCGTCAAGAT 475

AgCDC11A CCAGTTGACTATTATCGACACGCCGGGGTTCGGGGACTCGTTGGACAACTCCCCGTCCTT 533 AgCDC11B CCAGTTGACAATCATCGACACGCCGGGGTTCGGGGACTCGTTGTACAACTCGCCATCCTT 535

AgCDC11A CAACATGATCTCGGATTACATAAGGCACCAGTACGACGAAATCCTTTTGGAGGAGAGTCG 593 AgCDC11B TAACATGATCTCGGACTACATAAGGCACCAGTACGACGAAATCCTTCTGGAAGAGAGCCG 595 CDC11A/B-I1 AgCDC11A GGTGCGGCGGAACCCGCGGTTCAAGGACGGGCGCGTGCACTGCTGTCTCTACCTCATCAA 653 AgCDC11B CGTGCGGCGGAACCCGCGGTTCAAGGACGGGCGCGTGCACTGCTGTCTCTACCTCATCAA 655 CDC11A/B-I1 AgCDC11A TCCTACCGGGCACGGGCTTAAGGAAATCGACGTGGAGTTCATGAGGCAGCTCGGCCCGCT 713 AgCDC11B TCCGACCGGGCACGGGCTCAAGGAGATTGACGTGGAGTTCATGAGGCAGCTCGGCCCGCT 715

AgCDC11A TGTGAACGTGATTCCGGTGATCAGTAAGTCGGACTCGTTGACCCCGGATGAGTTAAAGCT 773 AgCDC11B CGTGAACGTGATTCCGGTGATCAGCAAGTCGGATTCGCTGACCCCGGACGAGCTGAAGCT 775

AgCDC11A CAACAAGAAGCTAATCATGGAGGATATTGACTACTACAACCTTCCGATCTATAGTTTTCC 833 AgCDC11B CAACAAGAAGCTAATAATGGAGGACATTGACTACTACAACCTTCCAATCTATAGTTTTCC 835

AgCDC11A GTTTGACCAGGACGTGGTGAGCGATGAGGACTACGAGACGAACACGTACTTGCGCTCGCT 893 AgCDC11B GTTTGACCAGGACGTGGTGAGCGATGAGGACTACGAGACGAACACGTACTTGCGCTCGCT 895

AgCDC11A ACTTCCTTTCTCGATCATTGGCTCCAATGAGACCTTCGAGACGGCTGAAGGTGGCGTTAT 953 AgCDC11B TCTTCCATTCTCGATTATTGGCTCCAACGAGACCTTCGAGGCTGCCGATGGCCGCGTCAT 955

AgCDC11A ACACGGACGCCGGTATCCTTGGGGGACCATAGATGTGGAGGACCCGGTGGTATCGGACTT 1013 AgCDC11B ACATGGGCGGCGGTATCCCTGGGGCACCGTGGACGTCGAGGACCCGGTGGTGTCGGACTT 1015

AgCDC11A CTGTGTTCTTCGGAATGCACTTTTGATTTCGCACTTGAACGACTTGAAGGACTACACCCA 1073 AgCDC11B CTGTGTCCTGCGGAACGCGCTCTTGATCTCACACCTGAATGACTTGAAAGACTACACTCA 1075

AgCDC11A TGAACTACTCTACGAACGCTACAGAACAGAGGCATTGTCTGGTGATATGCTGACTGCCAG 1133 AgCDC11B CGAGCTTCTCTACGAGCGCTACAGAACAGAAGCGTTGTCGGGTGATGTGCTGACAGCCAG 1135 AgCDC11A CAGTGTCTCGTCAAAGCTGATGAACAATGGGTCTACGGAATTTATCAGCTCACCAGCTCT 1193 AgCDC11B CAGCGTCCCATCTAAGACGGTGATTAACGGCTCAACAGAGTACGCCGGCTCGCCCGTGCC 1195 CDC11B-I2 AgCDC11A TTCCGGGACTGGCTCAGATTCAGCACGCGTGAGCGGCCAGGAGGCGGAGTCGCGCAACAG 1253 AgCDC11B ACCCGCTACGGCCTCGGACTCCGTGCATGTGAGCGGCCACGAGGCGGAGTCGCGCAACAG 1255

AgCDC11A CACGAAGCAGTCTTCTAACCAAGATACATACTTGGCACGGGAAGAGCAGATCAGGCTAGA 1313 AgCDC11B CACGAAGCAGTCTTCCAACCAGGATACGTACCTGGCGCGGGAGGAGCAGCTCAGACTGGA 1315

AgCDC11A GGAAAAGAGATTGAAGGCATTTGAAGAGCGTGTTCAGCAGGAATTGCTATCGAAGAGGCA 1373 AgCDC11B GGAGCAGAGATTGAAAGTGTTCGAGGAGCGCGTCCAGCAAGAACTGTTGTCGAAGAGGCA 1375

AgCDC11A AGAGTTATTACGGAGAGAGCAAGAATTAAGAGAGATCGAGGAACGTCTGGAAAAAGAAGC 1433 AgCDC11B AGAGCTATTGCGCAGAGAGCAAGAGTTGAGGGAGATCGAGGAGCGTCTGGAAAAAGAAGC 1435 CDC11A-I2 AgCDC11A CAAAACCAAGCAGGAAATTGAGGATTAAAATTCATTGATGTGACCTTTTACACGAAGCAA 1493 AgCDC11B CAAAA-CTA-GAGTCGAATGATGATAGAAAGT-ATTGGCGTGGTCTTTT--AC---GCAA 1487

AgCDC11A GAAAAGAATGGCTG-AAACATGTGAGCATCATACGCTAGTTAATTAACATGACATATTGC 1552 AgCDC11B AACAAGAGTAGCTGAAAACAGGTTAGCACCGCAAGCTAG-TAATCAACATGAC--A-T-C 1542

AgCDC11A GCGAAGGTATCGTGATATGTTCCAGTAAAAAATATTAACGTACCCA--TAATTCATTTAT 1610 AgCDC11B GCAGCGGTATCGTGACAT-ACCCAGTACAAGATTTTAACATACCCAGGCCATTCATTTGT 1601

AgCDC11A ACATATGCTAAGTAGGC-AACCGATAATACCACATG-CAA---GCAGCGAGAGGGACAGC 1665 AgCDC11B AGGTATGCTAAGTAGACAAAACGATAGTACC-CATGCCAGTGCGCGGGGTTTTGCATCGC 1660

AgCDC11A GATTCCTCGAGGTGATTCCACAATCATATTGATTTACTTCATCAAGACTCGCCTCGAGAA 1725 AgCDC11B GAGCCCT-AAAG-G---CGGCCATCAACTTGACTTAC-T--T----AA------GC-AA 1700 CDC11B-S2 AgCDC11A AGACACGGTTACGTAAAGTCAGAAATTAGAAAGGGAAAAAAAAAAAGCTGAGCTA-GTGT 1784 AgCDC11B AGTCCAGGCCTCGAGAA---A-AAATT-----GCACTATAAAAGCGGCGGGCATACGTGT 1751

AgCDC11A TAGTCTTCCAGGATGCCTAAGTACAGCCGCCGGCTAAAACGAAACCCAATATTGTATCAC 1844 AgCDC11B GAG-CATACCCGCTG-C-AAGAACAGCC-ACGGCGAGCAC-CCGGCCGCTGTTGCAGCGC 1806 CDC11A-S2 AgCDC11A ATAATTGGTA-TTCTGGAGTTTGGGCCTAGTAGGGATTCTACGTA-ATTCACCATCCGTA 1902 AgCDC11B AGGACGTCTAGGTGTCCAG-TAGGAACC-ATACGG---CCGCGTACACTCA-GGGCAGCA 1860 CDC11A-G4 CDC11B-G4 AgCDC11A ACATGC-GTTGACCATGTAC--CGATTTTCAGAGAA 1935 AgCDC11B GCTGCCGCTTCTTCGGGTACATCGAAATGACGAGGA 1896

Figure II.11. Pairwise alignment of the highly conserved AgCDC11A and AgCDC11B genes. Oligos are designed to bind in places with highest possible diversity. The open reading frames are marked with bold, colored letters (red: AgCDC11A, green: AgCDC11B). A ∆l∆t, actin Agcdc11A∆, actin Agcdc11B∆, actin

B ∆l∆t, chitin Agcdc11A∆, chitin Agcdc11B∆, chitin

Figure II.12. Deletion of AgCDC11A or AgCDC11B results in a partial or no septin phenotype. ∆l∆t (reference), Agcdc11A∆ (AgHPH17) and Agcdc11B∆ (AgHPH18) mycelia were grown for 12 h in liquid AFM at 30°C, fixed with formaldehyde and stained with Alexa-Phalloidin (A) or fixed with ethanol and stained with Calcofluor (B). Agcdc11A∆ mutants display morphological defects similar to the other septin null mutants, but neither actin nor chitin rings are missing. No phenotype of Agcdc11B∆ mutants has been found. Bars, 10 µm. Discussion

The filamentous ascomycete Ashbya gossypii 2001b). It has further been shown that the septin carries a Saccharomyces-like genome but its ring at the neck undergoes changes in protein morphogenesis differs markedly from budding dynamics to allow structural changes like single yeast. Large areas of nutrients are quickly ring to double ring transition, and that this is covered by A. gossypii mycelium through per- regulated through the phosphorylation state of manently polarized hyphal growth and branch the septins (Dobbelaere et al., 2003). In C. formation. Except during germination and in albicans, it has been speculated that sumolyation some mutants, isotropic growth is not observed. of the septins could be the bases for the different In stark contrast to budding yeast where nuclear patterns of septin structures (Martin and division is obligatory followed by cytokinesis and Konopka, 2004b). Thus, modification of the sep- cell separation (abscission), cytokinesis is not tins could be a key element in determining their observed during vegetative growth of A. organizational structure. The fact that we see a gossypii. It never propagates by yeast-like or great variety of different septin organizations in pseudohyphal growth like other filamentous A. gossypii itself also makes it unlikely that fungi. Nonetheless, A. gossypii is not only having bar-like rings in A. gossypii and continu- equipped with homologues to all S. cerevisiae ous rings in S. cerevisiae is solely the results of septins – the key players in cytokinesis, one is different septin proteins. The transient state of even duplicated (AgCDC11A, AgCDC11B). signal fading seen during single ring to double ring transition in A. gossypii is evidence for Cytokinesis is likely to be a crucial part of the profound structural rearrangements which likely sporulation program. Thus the A. gossypii comes along with changes in protein dynamics. septins could be required only late in the life Mutants of potential septin modification proteins cycle. However, our localization studies with could help to understand how such transitions AgSep7-GFP have revealed a complex set of are regulated and whether different structures structures that is already established in young are related to different modifications. FRAP mycelium, which makes it unlikely that the analysis could be used to analyze protein septins are only responsible for sporulation. A dynamics of individual structures. striking difference between AgSep7-GFP rings seen in A. gossypii and budding yeast is in their What is the nature of the septin bars we see in A. structural organization: whereas in S. cerevisiae gossypii? Do they represent filaments made from they are continuous, they are intermittent in A. individual septins? If so, it was possible that the gossypii, made from parallel bars. How can the gaps between the bars are in fact filled by the very same protein, expressed from the same other septins. All the septins together would then promoter, lead to such different structures in form a continuous ring. Our colocalization analy- different organisms? We must keep in mind that sis of AgCdc11p and AgSep7-GFP and in vitro we are not looking at all septins at the same interaction studies in budding yeast (Versele et time, but only at one single component of the al., 2004) rather support the hypothesis that each ring, AgSep7-GFP. In budding yeast, the septins filament incorporates all the septins, though. form heteropentamers with ScSep7p (ScShs1p) There is still the possibility that AgSep7-GFP being the most peripheral component of this bars and AgCdc11p bars just appear to co- structure (Versele et al., 2004). Presumably, the localize because they are very close together. role of Sep7 lies in recruiting other proteins to Ideally, we would tag all the septins and observe the septin ring, and not in defining the structural whether they together form continuous rings or organization of the ring itself. To investigate still bar-like rings. whether and which septins are crucial for the observed structural differences, one could re- How does this potential discontinuity of the rings place different subsets of septins in S. cerevisiae influence the recruitment of other proteins to with their A. gossypii homologues and see which these structures? AgHsl7-GFP colocalized with combinations lead to bar formation. There is also some but not all septin rings and actin rings were the possibility that the septin structure is not limited to mature, contracted septin structures. determined by the septins themselves. Discon- We have shown that even contracted septin tinuous rings are known from mating S. rings consist of bars. Thus, the bar-like organi- cerevisiae cells at the neck of shmoos (Ford and zation of septin rings in A. gossypii does not Pringle, 1991; Kim et al., 1991; Longtine et al., prevent recruitment of other proteins or even 1998; Cid et al., 2001a) or at the mother-bud- formation of continuous structures like actin neck of budding yeast cells bearing mutations of rings. Nevertheless, localization of actin rings or septin regulators (reviewed in (Gladfelter et al., AgHsl7-GFP to the asymmetric septin structures

CHAPTER II 26 at young branching sites or colocalization with gossypii septins during vegetative growth. We young, diffuse hyphal rings has never been have seen that the lack of septa in septin mu- observed. Possibly, hyphal septin rings need to tants causes the mycelium to be very susceptible mature before they can recruit other proteins. to hyphal damage. Whereas in wild type, dam- Alternatively, septin structures of different locali- age induced lysis is restricted to the affected zation and age may recruit different sets of hypha, it spreads over the entire mycelium in protein. septin mutants grown in liquid. Considering this, it is surprising that these mutants are able to Interestingly, we have seen that a diffuse portion form large colonies on solid medium at all. One of AgSep7-GFP always stays at the growing tips. can imagine that even without mechanical stress Do these faint, elongated filaments only serve as from shaking, lysis-inducing events should occur precursor for hyphal ring formation, or are they at some point during several days of growth. also involved in recruiting and/or maintaining Nonetheless, complete lysis of mature colonies actin patches at the tips? Deletion of single has never been observed. Mere size of the septin proteins (Agcdc3Δ, Agcdc10Δ and mycelium could prevent leakage of the entire Agcdc12Δ) leads to complete delocalization of cytoplasm. Especially when immobilized on solid AgSep7-GFP: neither distinct structures nor po- medium, sites of leakage should eventually be larization at the tips is seen in these mutants. clogged by escaping cytoplasm. Even so, the Although the actin cytoskeleton is difficult to amount of cytoplasm lost during such incidents stain in septin deletion mutants and actin could be enough to notably reduce growth patches are less clearly visible than in the refer- efficiency. ence strain or even reduced in number, actin patch polarization does not seem to be affected. Reduced radial growth rate is also likely linked to Even if the septins are not crucial for actin patch the morphological defects we have observed: A. polarity, they are essential for the formation of gossypii septin deletion mutants exhibit erratic actin rings, which are no longer observed in thickness of the hyphae, show difficulties in Agcdc3Δ and Agcdc10Δ, nicely fitting to the maintaining growth direction and have a disor- colocalization data. Agmyo1Δ mutants, which dered branching pattern. Regulation of hyphal likewise have no actin rings, are still able to form thickness may be required for optimal tip aberrant septa which allows limited sporulation. extension speed, whereas adjusting direction of The nature of this complementing mechanism in growth and the branching pattern should be A. gossypii has not yet been identified, but could critical to efficiently grow towards and exploit be similar to S. cerevisiae, where strains lacking regions rich in nutrients. How are the septins a contractile actomyosin ring can still undergo involved in these processes? It has been shown cytokinesis through formation of a septum by that the branching pattern of A. gossypii is vesicle fusion with the plasma membrane (Bi et largely determined by AgRax1p and AgRax2p. al., 1998; Lippincott and Li, 1998b; Vallen et al., AgBud10p is required for stable localization of 2000; Faty et al., 2002). Unlike in these AgRax2p at growing tips (Boudier, PhD thesis, Agmyo1Δ mutants, we have neither seen chitin 2005). Septin mutants in S. cerevisiae display rings nor septa in the above septin mutants. mislocalisation of ScBud3p, ScBud4p and Coming along with this is a total loss of sporula- ScBud10p and disruption of the axial budding tion capability. Thus, all pathways that can lead pattern (Flescher et al., 1993; Chant et al., to septum formation and sporulation depend on 1995). It is likely that the homologues of these the septins, be it actin ring formation or potential proteins are similarly localized by the septins in localized vesicle fusion. A. gossypii, which would explain why the branching pattern is disturbed in septin mutants. Notably, the severe or lethal phenotype of septin Strikingly, the septins at the branching site do not mutants in S. cerevisiae is largely due to the disappear after branch emergence but rather inability to complete cytokinesis in the absence stay there to form an asymmetric structure of yet of a proper septin ring at the bud neck (Hartwell, unknown function. AgHsl7p has never been 1971a). Contrasting budding yeast, cytokinesis found to localize to these places. Does this imply does not occur in A. gossypii mycelium before that the septins at the base of branches recruit a sporulation and hence disruption of cytokinesis different set of proteins than the hyphal rings? should not affect vegetative growth of this Are they required for stabilization of the angle fungus. Although septin deletion mutants in A. between the branch and the mother hyphae, thus gossypii are not lethal, they exhibit reduced helping to maintain direction of growth? Another radial growth speed and aberrant morphology. unanswered question is proposed by the faint but Together with the complex localization pattern permanent localization of septins to the growing which is already established in young mycelium, tips. This localization is not required for actin this clearly indicates significant roles of A. patch polarity, nevertheless maintenance of the

CHAPTER II 27 axis of polarity and hyphal diameter seems to be A very interesting aspect of the septins in A. affected in septin mutants. How may the septins gossypii is the duplication of CDC11. Despite contribute to polarity in A. gossypii? It has been 85 % identity between the two proteins, deletion shown that during isotropic bud growth, cortical of each results in some different phenotypes. components of the polarisome and the exocyst Whereas Agcdc11BΔ cannot be distinguished in S. cerevisiae are restricted to the bud mem- from the reference, Agcdc11AΔ strains display brane by a septin mediated membrane diffusion morphological defects that are typical for septin barrier (Barral et al., 2000; Takizawa and mutants. Strikingly, actin and chitin rings are still Morgan, 2000). Likewise there is evidence that formed in Agcdc11AΔ mycelia and sporulation is the septins might colocalize with sterols in C. not completely abolished but occurs at 30 % albicans and might establish or maintain efficiency. This indicates that altered morphology membrane polarity (Martin and Konopka, of small mutant mycelium is not the result of 2004a). In S. pombe, sterol localization corre- absent septa. It would be highly interesting to lates with sites of active cell growth and delete AgCDC11A in the AgSEP7-GFP back- cytokinesis, as it was revealed by Filipin staining ground to see which of the septin structures are (Wachtler et al., 2003). Are the septins in A. affected by this deletion. This has the potential to gossypii involved in maintaining cortical polarity answer many of the above questions by yielding at the growing tips? Lack of such a membrane insight into which structures are required for barrier could result in partial delocalization of which morphogenetic process. So far we could polarity factors over the entire hypha. This would not resolve the function of AgCdc11Bp. The two explain reduced growth and the swollen appear- proteins may be partially redundant, allowing ance of the hyphae in septin mutants. On the actin and chitin ring formation even if one of other hand, it is hard to imagine how the septins them is disrupted. This could be tested by ana- in A. gossypii could serve as barrier, considering lyzing double deletion mutants. Alternatively, the diffuse and bar-like appearance of septins at AgCdc11Bp could have adopted a function which growing tips. Additionally, Filipin does not only is of advantage in the natural environment of A. localize to growing tips in the reference strain but gossypii, but has so far escaped our obser- also in septin deletion mutants. Alternatively, the vations under laboratory conditions. We have septins could directly recruit and maintain polar- just begun to study these twins. More detailed ity factors at the tip. All the same, we cannot examination may reveal the processes in which completely abandon the hypothesis of cortical the two proteins are involved and why it was polarity. The space between the AgSep7-GFP or worthwhile for A. gossypii to preserve two copies AgCdc11p bars could be filled by other proteins of CDC11. that support the barrier function. Further, Filipin staining is prone to artifacts and does not reliably Thanks to the peculiar morphogenetic processes indicate the distribution of sterols in S. cerevisiae coming along with filamentous growth, future live cells (Valdez-Taubas and Pelham, 2003). studies of the septins in A. gossypii and other Localizing cortical exocytosis or polarity compo- filamentous fungi may draw secrets from these nents in the A. gossypii reference strain and versatile proteins, which uninucleated cells would septin mutants could shed light on the question have kept forever. of whether cortical polarity is also a relevant feature of apical growth.

CHAPTER II 28 Materials and Methods

A. gossypii methods, media and growth kers (McElver and Weber, 1992; Baudin et al., conditions 1993; Wach et al., 1994; Wendland et al., 2000). A. gossypii media, culturing, and transformation For G418 resistance, the deletion cassettes were protocols are described in (Wendland et al., amplified off the pGEN3 template using “gene 2000) and (Ayad-Durieux et al., 2000). A. name”-S1/S2 primer pairs. ClonNAT® resistance gossypii strains used here are listed in Table 2. was obtained using pUC19NATPS as template. To test the influence of high culture density, 10 – N*S1/N*S2 primer pairs were used for 20 ml of full medium were densely inoculated expression of the NAT1 resistance module under with spores and the cultures were grown until the control of the promoter and terminator of the vacuoles were clearly observable by phase targeted gene. NS1/NS2 primer pairs lead to contrast microscopy. For all other conditions, 50 NAT1 expression under the control of the – 100 ml of full medium were inoculated at low ScPDC1 promoter and terminator. Correct density, regularly controlled for the absence of integration was verified by analytical PCR with visible vacuoles and fresh medium was added 1 oligonucleotides “gene name”-(N)G1, -(N)G4, - – 2 hours before taking samples or exposing the I1/I2 (genomic) and G2.1, G3, V2PDC1P, cultures to conditions of potential stress. V3PDC1T, V2*NAT1, V3*NAT1 (marker). To Nocodazole stocks for the arrest and release exclude phenotypes by random mutations, at experiment were 3 mg/ml in DMSO and used at least two independent transformants were char- a final concentration of 15 μg/ml (Sigma). acterized for each deletion. Transformation of Hydroxyurea was directly added to the cultures multinucleate mycelium leads to heterokaryotic to a final concentration of 50 mM. Rapamycin cells, which contain a mixture of transformed and stocks (LC Laboratories, Woburn, MA) were wild-type nuclei, and usually do not display an 1 mg/ml in 90 % EtOH, 10% Tween-20 and used apparent phenotype. For subsequent analysis, at a final concentration of 200 nM (Loewith et al., homokaryotic mycelium was obtained by isola- 2002). To simulate the starvation response, low ting and growing single spores. To evaluate density cultures were washed twice and phenotypes of lethal mutants or mutants with resuspended in 40 mM MOPS [pH 7.0], 137 mM sporulation deficiency, spores from hetero- KCl. To test the lack of certain nutrients, different karyotic mycelium were germinated and media based on Ashbya Synthetic Dextrose analyzed under selective conditions. (ASD) medium were used: 1.70 g/l YNB w/o amino acids and w/o ammoniumsulfate (Difco), For the creation of the double deletion strains 0.69 g/l CSM-LEU (Bio 101), 1.00 g/l Asn, AgHPH29 and AgHPH31, at least three 0.01 g/l adenine, 1.00 g/l myo-inositol, 20 g/l homokaryotic isolates bearing the first deletion glucose, 50 mM MOPS [pH 6.5]. that were phenotypically not distinguishable were tested for correct integration of both ends of the Plasmid and strain construction deletion cassette and absence of the wild-type All DNA manipulations were carried out gene using verification PCR. One of these according to (Sambrook, 2001) with DH5αF’ as isolates was grown and transformed with the host (Hanahan, 1983). Plasmids used here are deletion cassette for the second gene, using a listed in Table 3. PCR was performed using different selection marker. If no homokaryotic standard methods with different polymerases mycelium of the first deletion mutant was from Roche (Basel, Switzerland), Amersham available yet (for creation of AgHPH27 and Biosciences (Little Chalfont, Buckinghamshire, AgHPH28), spores from heterokaryotic mycelium United Kingdom), or GenScript (Piscataway, NJ). were grown under selection and transformed Oligos were synthesized at MWG (Ebersberg, with the second deletion cassette. In both cases, Germany) or Microsynth (Switzerland) and all three primary transformants resulting from the restriction enzymes came from New England second transformation were isolated. Verification Biolabs or Roche. Oligonucleotide primers are PCR was used to test correct integration of both listed in Table 4. Plasmid isolation from yeast deletion markers. To test absence of both wild- and sequencing were performed as described in type genes, homokaryotic mycelium was isolated (Schmitz et al., 2006). from the double deletion mutants and verified using PCR. A. gossypii deletion mutants were generated using the PCR based one-step gene targeting To construct a strain with integrated histone-H4- approach with non-homologous selection mar- GFP (AgHPH004), the plasmid pAG-H4-GFP-

MATERIALS AND METHODS 29 KanMX6 (Alberti-Segui et al., 2001) was created using in vivo gap repair in S. cerevisiae. digested with BglII and SacI. Blunt ends were First, the plasmid pRS416CDC28GEN3 was generated using the 5’–3’ exonuclease activity of created by Amy Gladfelter: AgCDC28 was Vent polymerase. The 1,747-bp fragment was amplified from genomic DNA using oligos subcloned into pAIC (kindly provided by Philipp Cdc28aIEcoR1 and Cdc28aIIBamH1, with homo- Knechtle) opened at the ScaI site (Knechtle, logy upstream of Cdc28 including the promoter PhD thesis, 2002). The new plasmid pHPH001 region and limited downstream sequence. This carried a COOH-terminal fusion of GFP (S65T) PCR fragment was purified and digested with to the ORF of histone H4 (AgHHF1) with flanking EcoRI and BamHI, sites present in the oligos, to homologies to the AgADE2 locus. HHF1-GFP clone it into pRS316 that was opened with these was under the control of the AgHHF1 promoter same enzymes. The entire CDC28 ORF was and was terminated by ScADH1-T. 5 μg sequenced to confirm that PCR did not introduce pHPH001 were isolated from Escherichia coli, point mutations. pRS416CDC28GEN3 was cut digested with EcoRI–HindIII, and transformed with XhoI at two sites, one being 23 bp upstream into the partially deleted AgADE2 locus of the of Y18, the other being located in the pRS416 Agade2Δ1 strain (Knechtle, PhD thesis, 2002). backbone. The resulting fragments were cotrans- Transformants were obtained on minimal me- formed into the yeast strain DHD5 (Arvanitidis dium lacking adenine (ASD -ADE). and Heinisch, 1994) together with the annealed

oligonucleotides CDC28-Y18F-A / CDC28-- A C-terminal fusion of GFP to AgSEP7 was Y18F-B and CDC28-glue-A / CDC28-glue-B for obtained by amplifying pGUG and pGUC with in vivo recombination. The pair CDC28-Y18F-A / S7-G-S1 and S7-G-S2 or S7-G-S1 and CDC28-Y18F-B overlapped the first XhoI site S7-G-CS2, respectively. To generate AgSEP7- and contained the Y18F replacement flanked by GFP-NAT1, the pGUC cassette was first a total of 15 silent mutations to improve the constructed by digesting pGUG with BglII and fidelity of subsequent genomic integration into A. BamHI and ligating the gel purified 1026 bp gossypii. The second XhoI site was overlapped fragment into the dephosphorylated pUC19NATPS by the pair CDC28-glue-A / CDC28-glue-B, vector which was linearized with BamHI. The S7- which contained no mutations. The gap-repaired G–S1 primer was designed with homology to the plasmid (pAGHPH007) was isolated and verified C-terminal region of AgSEP7 before the stop by sequencing of the altered regions. codon. The PCR products were each trans- pAGHPH007 was digested with EcoRI, BamHI formed together with pAG4401, containing and PstI and transformed into A. gossypii ΔlΔt AgSEP7, into the yeast strain CEN.PK2 for in cells, resulting in AgHPH36. Correct integration vivo recombination. The resulting plasmids and presence of the altered region was verified pAGHPH002 and pAGHPH003 were purified by analytical PCR with the oligonucleotides and transformed into A. gossypii ΔlΔt cells, CDC28-IB / CDC28-Y18F-IA (binds to altered leading to the strains AgHPH05 and AgHPH06. sequence, only), G3 / CDC28-G4.2 and CDC28- For integration into the genome, the two IB / CDC28-IA-Y18 (binds to wild-type sequence, constructs were first subcloned into pUC19 to only). For additional verification, a fragment con- eliminate ARS activity. pUC19 was opened with taining the altered sequence was amplified using SalI and XbaI, AgSEP7-GFP-GEN3 was excised CDC28-G1.2 / CDC28-IB and digested with from pAGHPH002 with EcoRI and SphI and KpnI, which cuts the mutated fragment at two pAGHPH003 was cut with XhoI and SpeI to places and the wild-type fragment and one site, extract AgSEP7-GFP-NAT1. The fragments only (Appendix 1.8). were gel purified and separately ligated into the dephosphorylated pUC19 fragment to produce The plasmid carrying AgSWE1 (pAGHPH004) pUCHPH002 and pUCHPH003. The cassettes was generated by amplification of the gene from were excised with EcoRI and transformed into A. genomic A. gossypii DNA using the primers gossypii ΔlΔt cells, giving rise to AgHPH07 and SWE1-B1 and SWE1-B2, which contain EcoRI AgHPH08. and BamHI restriction sites. The fragments were purified, cut and ligatet into pRS416. The result- To generate ASG41 (AgHSL7-GFP-GEN3, con- ing plasmid was verified by sequencing. structed by Amy Gladfelter), the oligos Hsl7-S1 and Hsl7-S2 (with homology to the C-terminus of To C-terminally tag SWE1 with GFP or 13-myc, AgHSL7) were used with the template pGUG to the pGUC or pAG13-myc cassette was amplified generate PCR product which was used to with SWE1-G-S1 and SWE1-G-NS2 or SWE1- directly transform A. gossypii cells. MS1 and SWE1-MS2, respectively. The resulting PCR products were cotransformed into the yeast To make the nonphosphorylatable Y18F muta- strain DHD5 together with pAGHPH004 for in tion of AgCDC28, the plasmid pAGHPH007 was vivo recombination, giving rise to pAGHPH005

MATERIALS AND METHODS 30 and pAGHPH006. Correct fusion was verified by containing 1 mM Na3VO4 and 1 mM β-glyerol- sequencing. Transformation of the plasmid phosphate and suspended in ice-cold lysis buffer pAGHPH006 into A. gossypii ΔlΔt cells produced (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM the strain AgHPH34. To obtain AgHPH32, EDTA, 1% NP-40, 2 mM sodium pyrophosphate, pAGHPH005 was digested with BmgBI and 1 mM Na3VO4, 1 mM β-glyerolphosphate and BssHII before transforming it into A. gossypii ΔlΔt Roche complete protease inhibitor cocktail). An cells, leading to genomic integration. equal volume of 0.5 mm glass beads was added to this suspension, and cells were broken by For overexpression of AgSWE1, the plasmid vigorous vortexing during 6 x 30 sec intervals in pAGHPH008 was constructed: the ScHIS3 a bead beater at 4 °C. Total cellular proteins promoter was amplified off pAGrPXC (kindly were separated by SDS-PAGE (12%) and provided by Dominic Hœpfner) using the primer transferred to nitrocellulose membranes (Amers- pair MH1 and SWE1-SH2. The GEN3 cassette ham Biosciences), using standard conditions. was amplified using the oligonucleotides MH2 Blocking was performed with 5% milk in TBS- and SWE1-SH2 with pGEN3 as template. MH1 Tween-20 (0.1%). For detection of phosphory- and MH2 contained homology to both, the GEN3 lated Cdc28p, rabbit anti-phospho-cdc2(Tyr15) cassette and the ScHIS3 promoter, generating (Cell Signaling, #9111) was used at 1:1000 in an overlap between the two PCR products. TBS, 5% BSA, 0.1% Tween-20, and HRP-anti- SWE1-SH1 and SWE1-SH2 added 45 bp homo- rabbit (Cell Signaling, #7074) was used at logy to a region upstream of AgSWE1 START 1:2000 in TBS, 5% milk, 0.1% Tween-20. As and 54 bp homology to the very START of positive control for phosphorylated CDK, total AgSWE1. The two PCR products were used cellular proteins were extracted from the yeast together as template in a subsequent PCR strain DLY5544 (cdc12-6, GAL/SWE1), grown in reaction with the primers SWE1-SH1 and SWE1- YP 2 % sucrose and induced by adding 2 % SH2. The resulting GEN3-ScHIS3 cassette was galactose. For detection of HA tagged proteins, fused in front of AgSWE1 by in vivo recom- mouse anti-HA was used at 1:1000 in TBS, 5% bination with pAGHPH004 in the yeast strain BSA, 0.1% Tween-20, and HRP-anti-mouse DHD5, leading to pAGHPH008. The plasmid (Jackson Immunoresearch) was used at 1:1000 was purified and verified by sequencing and in TBS, 5% milk, 0.1% Tween-20. Detection of transformed into A. gossypii ΔlΔt cells to produce labeled proteins was performed by using the AgHPH37. To C-terminally tag GEN3-PScHIS3- enhanced chemiluminescence (ECL) detection AgSWE1 with HA, a 980 bp fragment carrying system (Amersham Biosciences). Either non- the carboxy end of AgSWE1 fused to 6HA was specific cross reacting bands or bands from cut out of pAgSWE1-HA-NAT1 using MscI and ponceau red staining of the membrane were ScaI. pAGHPH008 was opened with MscI and used as loading controls. XbaI. T4 DNA polymerase was used to fill the 5´ protruding end generated by XbaI. The dephos- Fluorescence stainings, image acquisition phorylated 8910 bp vector fragment was gel and processing purified and ligated with the 980 bp insert to DNA stainings (Hoechst 33342) and anti-tubulin produce pAGHPH009. In frame fusion of the tag immunofluorescence stainings were performed was verified by sequencing. Transformation of as described previously (Gladfelter et al., 2006). the plasmid pAGHPH009 into A. gossypii ΔlΔt Rabbit anti-ScCdc11p (Santa Cruz Biotechno- cells produced the strain AgHPH38. To generate logy) was diluted in PBS and used at a 1/10 pAgSWE1-HA-NAT1 (done by Amy Gladfelter), dilution. Rabbit anti-phospho-cdc2(Tyr15) was the Swe1F1 and Swe1F2 oligos, which have used at 1/50 for immunofluorescence. For in vivo homology to the C-terminus of AgSWE1 and the observation of mycelium containing GFP tagged universal tagging cassette, were used in a PCR proteins, the cells were grown for 10 - 12 hours reaction with the pN1-6HA (pAGT105-kindly in full medium and then washed and resus- provided by Andreas Kaufman) template and the pended in ASD or MOPS/KCl for observation resulting product was cotransformed into yeast under the microscope. cells with the plasmid pAGHPH004. Recombi- nation of the plasmid with the linear PCR Visualization of the actin cytoskeleton by Alexa fragment resulted in yeast cells resistant to Fluor Phalloidin 488 was done as described in CloNAT. Plasmids were rescued from these (Knechtle et al., 2003) with slight modifications: resistant yeast strains and correct fusion of 6HA to improve the yield when staining young to AgSWE1 was verified by restriction digest. mycelia, 9 ml of culture were mixed with 1 ml of 37 % formaldehyde and 10 μl of 10 % Triton-X Protein extraction and Western blotting and fixed for 10 min. Mycelia were collected in Cells grown under the desired conditions were 15 ml Falcon tubes by 1 min centrifugation in a collected, washed once with ice-cold PBS Heraeus Minifuge at 1000 rpm and resuspended

MATERIALS AND METHODS 31 in 900 μl PBS containing 4 % formaldehyde and The microscopy setup used was the same as processed as described previously. For staining described in (Hoepfner et al., 2000; Knechtle et of the actin cytoskeleton while preserving the al., 2003; Gladfelter et al., 2006). The following GFP signal mild fixation was used: The mycelia filter sets were used for the different fluoro- were fixed 20 min in 2 – 4 % p-formaldehyde, phores: #02 for Hoechst, Calcofluor and Filipin, washed twice with PBS and resuspended in 100 #10 for Alexa 488, #15 for rhodamine 568 (Carl μl PBS containing 0.6 μM Rhodamine-Phalloidin. Zeiss) and #4108 for GFP (Chroma Technology). After incubating the cells for one hour on ice, they were washed five times with PBS, resus- Multiple planes (20 – 50 for still pictures and 3 – pended in 50 – 200 μl mounting medium and 7 for time-lapse pictures) with a distance mounted on slide (Sambrook, 2001; Knechtle, between 0.3 and 0.5 μm in the Z-axis were PhD thesis, 2002; Laissue, PhD thesis, 2004), taken. MetaMorph 4.6r9 software (Universal Andreas Kaufmann, personal communication). Imaging Corp) was used for “no-neighbors” 2D- deconvolution. The stacks were flattened into a Calcofluor white was used to stain chitin rings. single plane using stack arithmetic “maximize” 20 μl of a 1 mg/ml Calcofluor solution were command. Outlines of the cells were obtained by added to 100 μl of liquid culture, incubated for 5 doing phase-contrast Z-series. The stacks were passed through the Metamorph filter “detect min, washed twice with dH2O and mounted on a glass slide for microscopic inspections. edges: Laplace 2” and flattened into a single plane using the stack arithmetic “sum” command. To visualize the distribution of sterols, a fresh Fluorescent and phase-contrast images were stock of 2 mg/ml Filipin in DMSO was prepared. scaled and overlayed using “color align”. 2 - 4 μl of this solution were added to 2 ml liquid culture (2 μg/ml final concentration) and incu- Time-lapse series were taken as described in bated for 2 min. During the incubation time, the (Gladfelter et al., 2006) and assembled into mycelia were washed twice with dH O, once with movies using QuickTimePro 6.5. Mitotic events 2 in Movie 1 were labeled manually using Adobe AFM and resuspended in 500 μl AFM. The toxic Photoshop CS. effect of Filipin resulted in rapid death of the mycelia, necessitating immediate microscopic inspection.

MATERIALS AND METHODS 32 Table 2. A. gossypii strains used in this study

Strain Relevant Genotype Source

Reference (ΔlΔt) leu2Δ thr4Δ (Altmann-Johl and Philippsen, 1996); Mohr 1997 Agade2Δ1 Agade2(310-566)Δ::GEN3, leu2Δ thr4Δ Knechtle, thesis, 2002

AgH4-GFP pAG-H4-GFP-KanMX6[AgHHF1-GFP-GEN3], leu2Δ thr4Δ (Alberti-Segui et al., 2001) AgHPH04 AgHHF1, AgADE2_AgHHF1-GFP, leu2Δ thr4Δ, G418s this study AgHPH05 pAGHPH002[AgSEP7-GFP-GEN3], leu2Δ thr4Δ this study AgHPH06 pAGHPH003[AgSEP7-GFP-NAT1], leu2Δ thr4Δ this study AgHPH07 AgSEP7-GFP-GEN3, leu2Δ thr4Δ this study AgHPH08 AgSEP7-GFP-NAT1, leu2Δ thr4Δ this study AgHPH09 pAG-H4-GFP-KanMX6[AgHHF1-GFP-GEN3], AgSEP7-GFP-NAT1, leu2Δ thr4Δ this study AgHPH10 Agcdc3Δ::GEN3, AgSEP7-GFP-NAT1, leu2Δ thr4Δ this study AgHPH14 Agcdc10Δ::GEN3, Ag SEP7-GFP-NAT1, leu2Δ thr4Δ this study AgHPH15 Agcdc12Δ::GEN3, AgSEP7-GFP-NAT1, leu2Δ thr4Δ this study Cdc42Q61H AgCDC42, Agcdc42G183C, thr4Δ Köhli, diploma thesis, 2003 AgHPH16 AgSEP7-GFP-NAT1, AgCDC42, Agcdc42G183C, thr4Δ this study hsl1ΔNAT1 Aghsl1Δ::NAT1, leu2Δ thr4Δ this study, A Gladfelter AgHPH17 Agcdc11AΔ::GEN3, leu2Δ thr4Δ this study AgHPH18 Agcdc11BΔ::GEN3, leu2Δ thr4Δ this study ASG41 AgHSL7-GFP-GEN3, leu2Δ thr4Δ this study, A Gladfelter AgHPH19 Agcdc12Δ::NAT1, AgHSL7-GFP-GEN3, leu2Δ thr4Δ this study AgHPH20 Aghsl1Δ::NAT1, AgHSL7-GFP-GEN3, leu2Δ thr4Δ this study AgHPH21 Aghsl7Δ::GEN3, leu2Δ thr4Δ this study AgHPH22 Agmih1Δ::NAT1, leu2Δ thr4Δ this study AgHPH23 Agmih1Δ::GEN3, leu2Δ thr4Δ this study ASG35 Agswe1Δ::NAT1, leu2Δ thr4Δ this study, A Gladfelter AgHPH24 Agswe1Δ::GEN3, leu2Δ thr4Δ this study AgHPH25 Agcdc3Δ::GEN3, Agmih1Δ::NAT1, leu2Δ thr4Δ this study AgHPH26 Agcdc10Δ::GEN3, Agmih1Δ::NAT1, leu2Δ thr4Δ this study AgHPH27 Agcdc12Δ::GEN3, Agmih1Δ::NAT1, leu2Δ thr4Δ this study AgHPH28 Aghsl1Δ::NAT1, Agmih1Δ::GEN3, leu2Δ thr4Δ this study AgHPH29 Aghsl1Δ::GEN3, Agmih1Δ::NAT1, leu2Δ thr4Δ this study AgHPH30 Aghsl7Δ::GEN3, Agmih1Δ::NAT1, leu2Δ thr4Δ this study AgHPH31 Agswe1Δ::GEN3, Agmih1Δ::NAT1, leu2Δ thr4Δ this study AgHPH32 AgSWE1-13myc-GEN3, leu2Δ thr4Δ this study AgHPH34 pAGHPH006[AgSWE1-GFP-NAT1], leu2Δ thr4Δ this study AgHPH35 Agcdc28Δ::NAT1, leu2Δ thr4Δ this study AgHPH36 Agcdc28Y18F-GEN3, leu2Δ thr4Δ this study AgHPH37 pAGHPH008[GEN3-PScHIS3-AgSWE1], leu2Δ thr4Δ this study AgHPH38 pAGHPH009[GEN3-PScHIS3-AgSWE1-6HA], leu2Δ thr4Δ this study AgHPH39 pAGSWE1HANAT1 [AgSWE1-6HA-NAT1], leu2Δ thr4Δ this study With the exception of plasmidic strains (square brackets), all analyzed mycelia were homokaryotic (all nuclei have same genotype). This was obtained either by sporulating homokaryotic strains or by applying selective pressure during germination.

MATERIALS AND METHODS 33 Table 3. Plasmids used in this study

Plasmid Vector Relevant insert Source pUC19 ––– ––– pRS416 ––– ––– (Sikorski and Hieter, 1989) pGEN3 pAF100 GEN3 (Wendland et al., 2000) pAg13-myc pGEN3 13myc-GEN3 (Gladfelter et al., 2006) pGUG pGEN3 GFP-GEN3 (Knechtle et al., 2003) pUC19NATPS pUC19 NAT1 Hœpfner, personal communication pGUC pGUG GFP-NAT1 this study pAIC pBSIISK(+)Sca- AIC (partial AgADE2) Knechtle, Thesis, 2002 pAG-H4-GFP-KanMX6 pAG1552 AgHHF1-GFP-GEN3 (Alberti-Segui et al., 2001) pHPH001 pAIC AgHHF1-GFP-GEN3 this study pAG4401 pRS416 AgSEP7 Mohr, Thesis, 1995; Dietrich et al., 2004 pAGHPH002 pAG4401 AgSEP7-GFP-GEN3 this study pAGHPH003 pAG4401 AgSEP7-GFP-NAT1 this study pUCHPH002 pUC19 AgSEP7-GFP-GEN3 this study pUCHPH003 pUC19 AgSEP7-GFP-NAT1 this study pAGHPH004 pRS416 AgSWE1 this study pAGHPH005 pAGHPH004 AgSWE1-13myc-GEN3 this study pAGHPH006 pAGHPH004 AgSWE1-GFP-NAT1 this study pRS416CDC28GEN3 pRS416 AgCDC28-GEN3 this study, A Gladfelter pAGHPH007 pRS416Cdc28GEN3 Agcdc28Y18F-GEN3 this study pAGrPXC YCplac111 PScHIS3-CFP Hœpfner, personal communication pAGHPH008 pAGHPH004 GEN3-PScHIS3-AgSWE1 this study pAGSWE1HANAT1 pAGHPH004 AgSWE1-6HA-NAT1 this study, A Gladfelter pAGHPH009 pAGHPH008 GEN3- PScHIS3-AgSWE1-6HA this study pAG plasmids are based on the pRS416 vector Sikorski and Hieter, 1989.

MATERIALS AND METHODS 34 Table 4. Oligonucleotide primers used in this study

Primer Sequence 5'-3' S7-G-S1 CAATTTGCTAGTGATGGATATGCTTCCCGCGCAACAGGCCAGGTACAAggtgcaggcgctggagctg S7-G-S2 GCATTGACCCTCGGGCAAAGGATAAGATATATAGTTAGAAGATGTTagggacctggcacggagc S7-G-CS2 GCATTGACCCTCGGGCAAAGGATAAGATATATAGTTAGAAGATGTTgattacgccaagcttgcatgcc S7-G-G1 GCCAAACAACAAAAGCTGGAG S7-G-G4 GTGCCCAGCATTTCATGGAAC S7-G-CG4 GCCGTACGCGATCTGGCAATAGATGCGACATCC Hsl7-S1 GACAAATACCACTGAGTTACACAATATTGGCGGTCATGCCTACATGATTAAATTGggtgcaggcgctggagctg Hsl7-S2 GTATGCGGAAGCATAAATAGAAATGTTGCTTTTATGTTAGCATAGCGCGAagggacctggcacggagc C3-S1 GCGTTATACAGGACCTACACACGTACAGGACTACCAAATCAGAACAAGCgctagggataacagggtaat C3-S2 GTGACAATAATCAAGACAAATAGACTGTTTTTTTCGATTGCGGGTAGCaggcatgcaagcttagatct C3-G1 AAACAGCGGACCACCACAAC C3-G4 GACTTCTGCTGGCGGATACC C3-I1 CCCACGCAGAGCAAATTGAG C10-S1 GTTCACTCAAAGGAGCGCAAACAGCACTACTCATCCGAAACACGTTTACACCAGTTCCgctagggataacagggtaat C10-S2 GTTTGAGTACCCTCAATATTGGTAGATCTGGTGACTTTTAAAAAGTTAGGGGTTATTCaggcatgcaagcttagatct C10-G1 CTCCGCCTCCTTAGGAACTG C10-G4 TGTCGCTTACTGCAGGCCATC C10-I1 GTGTCCACGATGTGCTTCTC CDC11A-S1 CATAAGACGCAAACAGCATGTCCAGTATCATCGAGGCCTCAGCCGCGgctagggataacagggtaat CDC11A-S2 CCCTACTAGGCCCAAACTCCAGAATACCAATTATGTGATACAATATTGGGaggcatgcaagcttagatct CDC11A-G1 TTCGACGAGCAGCCGAACAG CDC11A-G4 CGGTACATGGTCAACGCATGTTACG CDC11A/B-I1 TTGAACCGCGGGTTCC CDC11A-I2 CCAAGCAGGAAATTGAGGAT CDC11B-S1 GATAAGACACAGTACAATGGCGGGCATTATTGAAGCGTCAGTAGCAgctagggataacagggtaat CDC11B-S2 CTTAAGTAAGTCAAGTTGATGGCCGCCTTTAGGGCTCGCGATGCAAaggcatgcaagcttagatct CDC11B-G1 GCGACAGATCCTGTAATGC CDC11B-G4 GCCGTATGGTTCCTACTGGACAC CDC11B-I2 TAACGGCTCAACAGAGTACGC C12-S1 CCTCAGACGCAAGGGCAACAGTGCAATAGGTGTGTTTGGTAACTTCgctagggataacagggtaat C12-S2 GCTGATCTGTCATTTCTAGTTGGTTCCACTACTGAAGATGATACGGaggcatgcaagcttagatct C12-NS1 CCTCAGACGCAAGGGCAACAGTGCAATAGGTGTGTTTGGTAACTTCccagtgaattcgagctcgg C12-NS2 GCTGATCTGTCATTTCTAGTTGGTTCCACTACTGAAGATGATACGGtacgccaagcttgcatgcct C12-G1 GATTATGTCCGCGTCGCAC C12-G1.2 GCGGCTTCCAAAGGACAAC C12-G4 TGGCACGGCTGCATCTTTAC C12-I1 GCTGCCTCCGATTCTGATTC HSL1-NS1 GCAGCACAGAGTGTAAGAAACCACAGAGGAGGATGACCGCAGGGCAccagtgaattcgagctcgg HSL1-NS2 CATTTGCAGCATTCTCCTTTGCTTTAATGAGTGCCTCAATGTCTGtacgccaagcttgcatgcct HSL1-NG1 GCTACTATTGAAGGTTGCAGC HSL1-NG4 CTTCTTATGTCGGCGTACCTAGTG HSL1-I2 CGCAGCAACCAAGGTGTCTG HSL1-S1.2 GTGTAAGAAACCACAGAGGAGGATGACCGCAGGGCACCGAGACGAGAGgctagggataacagggtaat HSL1-S2.2 CTCATCACTTCTTCCCTTTTGCTTGACAGACACCTTGGTTGCTGCGCTATaggcatgcaagcttagatct HSL1-G1.2 TGCAGCCCGTTTCCAACTAC HSL1-S4.2 GAGTGCCTCAATGTCTGTG HSL1-IA TTGCGAGGGCCTCTAACAAC HSL1-IB TCTCCGCCATGTGAATCAGC HSL7-S1.2 GTATCAAGGGCCACGGGGTGCTAGAAGGCGCTGCGTACCGGGAGAAGTACgctagggataacagggtaat HSL7-S2.2 CGGTCGGAAGTGATCATGAGAAGTGAAATTCAGTTGCCACAGTGCTAGCCaggcatgcaagcttagatct HSL7-G1.2 GTTTGTTTACTGCGGTGGTC HSL7-G4.2 GTTAGCATAGCGCGAACA HSL7-I2.2 ACCAGGTCGAAGAAACAC MIH1-S1 CAAAAAAAAGCCTACTTTCTCTACTTAATAGTGTTTACTCGAGCACgctagggataacagggtaat MIH1-S2 CTTAAGAGTCGTTGTCCTCCTCCTTGACGATCTCTGGGAAAAGCCaggcatgcaagcttagatct MIH1-NS1 CAAAAAAAAGCCTACTTTCTCTACTTAATAGTGTTTACTCGAGCACccagtgaattcgagctcgg MIH1-NS2 CTTAAGAGTCGTTGTCCTCCTCCTTGACGATCTCTGGGAAAAGCCtacgccaagcttgcatgcct MIH1-G1 CAGGCGCAGAAGTCGTTAC MIH1-G4 GACGCGCAGTTGTTGCTTC MIH1-I1 CCTCTGTCTCGCTCTTTCG SWE1-NS1 CAAGGTACCAGCGGCAAGCGCGCAGAGAAGACACAGACTAAGAATGccagtgaattcgagctcgg SWE1-NS2 CCTTATGGTGGCCGGGGAGCGCGATATGCGCCGAACATGCATTCTGtacgccaagcttgcatgcct SWE1-S1 CAAGGTACCAGCGGCAAGCGCGCAGAGAAGACACAGACTAAGAATGgctagggataacagggtaat SWE1-S2 CCTTATGGTGGCCGGGGAGCGCGATATGCGCCGAACATGCATTCTGaggcatgcaagcttagatct SWE1-B1 gcgaattcCGCCTCAGCTTGATCATCGTCAC SWE1-B2 cgggatccATCGGTATCTGCCGAACACAATGC SWE1-G-S1 CCATCATCCAAGAAGACGACTTTGGTCCCAAGCCGGAGTTCTTCAGCggtgcaggcgctggagctg SWE1-G-NS2 CGCACGTTCACTTGTAGCCGAGAAGCCCCTTATGGTGGCCGGGGAGgattacgccaagcttgcatgcc SWE1-MS1 CCATCATCCAAGAAGACGACTTTGGTCCCAAGCCGGAGTTCTTCAGCcggatccccgggttaattaacggtgaa SWE1-MS2 CGCACGTTCACTTGTAGCCGAGAAGCCCCTTATGGTGGCCGGGGAGcactagtggatctgatatcagatctgg AgSWE1 G1 CTCACCTCGAGGCCACTCCGTGTGAGG

MATERIALS AND METHODS 35 SWE1-G-G1 CATCGCGCCGGAAATCATC AgSWE1 G4 GCAGAGACGCTCAGAGAGATTCCCATTG AgSWE1 I GCGACTGAGAGCGATCCGATATTAAGG SWE1-SH1 AGGAGAAGGGCATACAGTTCCCCACGAATTTCGGCATAGAAAGGCgaattcaggcatgcaagcttagatct SWE1-SH2 GTTCAGAAACGGCGACTCGCGCAGCGCTGCCATGCTGTCGTGCCGGAGCGTCATggatccccgggtaccgagctctttg MH1 GTATTACCCTGTTATCCCTAGCATTCTATTACTCTTGGCCTCC MH2 GGAGGCCAAGAGTAATAGAATGCTAGGGATAACAGGGTAATAC HISP-HG2 TTTCCACCTAGCGGATGAC SWE1-HG1 ATGGACGCCACAGGTTATCTC CDC28-N*S1 ACTCCCTATGTACCCCACGGGCACGGGCCCATAAACCGGCAAAAAACGCGCCTGCGACAGA AAATTTTGTGTACCTCTACCACCAGCACCCTCatgggtaccactcttgacgac CDC28-N*S2 AGCAGCGCAATCTACATGCAAACGCGGTACAGCACCAGCGCTACTATTTATACCACCTACG TAGCTGCCTGTTCTGGCAGCGAGCGGGCCCTAggggcagggcatgctcatgtag CDC28-N*G1 TGCTGACGATGGGCTCCTTC CDC28-N*G4 TGTGGACCCTCCCAGTTACC CDC28-IB TGCACATTTCGGCGAAGATG CDC28-G1.2 AGGAGTGACCTCCTCTACAG CDC28-IA-Y18 GAACGTATGGTGTCGTCTAC CDC28-Y18F-A CCCTCATGAGCGATCTCACCAACTACAAGCGCTTGGAAAAAGTCGGTGAAGGTACCTTCGG GGTTGTTTATAAAGCGGTTG CDC28-Y18F-B CGCCACGATGCGCTGCCCATGCCGCAGGTCAACCGCTTTATAAACAACCCCGAAGGTACCT TCACCGACTTTTTCCAAGC CDC28-glue-A GGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGC CDC28-glue-B GCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCC CDC28-Y18F-IA GGTACCTTCGGGGTTGTTTA CDC28-G4.2 ATGTGCGCGAGGCTCTTTC Cdc28aIEcoR1 CATAGAATTCACTTCCGTGCCTGGGTGCAGCAGCATCGGCTTC Cdc28aIIBamH1 GTTCGGATCCTGACGATGGGCTCCTTCGACGGCCTGTTG Swe1F1 CCATCATCCAAGAAGACGACTTTGGTCCCAAGCCGGAGTTCTTCAGCAAAACGACGGCCAGTGAATTCG Swe1F2 CGCACGTTCACTTGTAGCCGAGAAGCCCCTTATGGTGGCCGGGGAGACCATGATTACGCCAAGCTTGC G2.1 tgcctccagcatagtcgaag G3 tcgcagaccgataccaggatc V2PDC1P gaacaaacccaaatctgattgcaaggagagtgaaagagcctt V3PDC1T gaccagacaagaagttgccgacagtctgttgaattggcctg V2*NAT1 gtgtcgtcaagagtggtacc V3*NAT1 acatgagcatgccctgcccc GG2 catcaccttcaccctctccac MYCI cgagtccgttcaag tcttcttctgag

Lower case letters are regions of homology to the cassette containing a selectable marker. Underlined letters represent intragenic replacements (alternative codons, point mutations).

MATERIALS AND METHODS 36 Appendix

I. Verification PCRs

II. Plasmid maps

III. Sequence alignments

APPENDIX 37 A

G1 G3

ScTEF2 ScTEF2 P GEN3 T

G2.1 G2 G4

G1 V3*NAT1 V3PDC1T

ScPDC1 ScPDC1 P ClonNAT1 T

V2PDC1P V2*NAT1 G4

G1 I2

P ORF T

I1 G4

Appendix 1.1. Oligos used for verification of deletion strains. (A) Verification of correct integration of the GEN3 deletion cassette. (B) Verification of correct integration of the ClonNAT deletion cassette. (C) Verification of absence of the targeted ORF in homokaryons. PCR products are obtained in wild type and heterokaryons, only. AgHPH22 (Agmih1∆::NAT1) AgHPH23 (Agmih1∆::GEN3) A

1 2 3 4 5 6 7 8 9 10 11 12

# 5 # 7 # 14 # 3 # 8 # 14 heterok. heterok. heterok. heterok. heterok. heterok.

1, 3, 5: MIH1-G1 + V2PDC1P → 506 bp 2, 4, 6: MIH1-G4 + V3PDC1T → 401 bp 7, 9, 11: MIH1-G1 + G2.1 → 508 bp 8, 10, 12: MIH1-G4 + G3 → 618 bp

AgHPH22 (Agmih1∆::NAT1) wild-type allele B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

# 5A # 5C # 14F # 5 homokaryon homokaryon homokaryon heterokaryon

1, 4, 7, 10, 13: MIH1-G1 + V2PDC1P → 506 bp 2, 5, 8, 11, 14: MIH1-G4 + V3PDC1T → 401 bp 3, 6, 9, 12, 15: MIH1-G1 + MIH1-I1 → 503 bp (wt)

Appendix 1.2. Verfification of Agmih1∆ strains. (A) heterokaryons (B) homokaryons AgHPH26 AgHPH27 (Agcdc10∆::GEN3, (Agcdc12∆::GEN3, AgHPH25 (Agcdc3∆::GEN3, Agmih1∆::NAT1) Agmih1∆::NAT1) Agmih1∆::NAT1) A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

# 1 # 3 # 9 # 12 # 1 # 4 # 7 heterok. heterok. heterok. heterok. heterok. heterok. heterok.

1, 3, 5, 7, 9: C3-G1 + G2.1 → 530 bp 2, 4, 6, 8, 10: C3-G4 + G3 → 615 bp 11: C10-G1 + G2.1 → 511 bp 12: C10-G4 + G3 → 621 bp 13, 15, 17, 19: C12-G1.2 + G2.1 → 512 bp 14, 16, 18, 20: C12-G4 + G3 → 587 bp

AgHPH25 AgHPH26 AgHPH27 (Agcdc3∆::GEN3, (Agcdc10∆::GEN3, (Agcdc12∆::GEN3, Agmih1∆::NAT1) Agmih1∆::NAT1) wild-type allele Agmih1∆::NAT1) B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

# 1 # 3 # 9 # 3 # 12 # 1 # 4 # 7 heterok. heterok. heterok. heterok. heterok. heterok. heterok. heterok. (Agcdc3∆) (Agcdc3∆) (Agcdc3∆) (Agcdc10∆) (Agcdc10∆) (Agcdc12∆) (Agcdc12∆) (Agcdc12∆)

1, 4, 7, 10, 13, 16, 19, 22, 25: MIH1-G1 + V2PDC1P → 506 bp 2, 5, 8, 11, 14, 17, 20, 23, 26: MIH1-G4 + V3PDC1T → 401 bp 3, 6, 9, 12, 15, 18, 21, 24, 27: MIH1-G1 + MIH1-I1 → 503 bp (wt)

Appendix 1.3. Verfification of Agseptin∆mih1∆ strains. (A) Verification of the heterokaryotic septin knock-outs. (B) Verfification of the homokaryotic Agmih1∆ background. AgHPH27 (Agcdc12∆::GEN3, Agmih1∆::NAT1) wild-type allele A

1 2 3 4 5 6 7 8 9 10 11 12

# 1A # 4C homokaryon homokaryon 1, 4, 7, 10: C12-G1.2 + G2.1 → 512 bp 2, 5, 8, 11: C12-G4 + G3 → 587 bp 3, 6, 9, 12: C12-G1.2 + C12-I1 → 336 bp (wt)

AgHPH27 (Agcdc12∆::GEN3, Agmih1∆::NAT1) wild-type allele B

1 2 3 4 5 6 7 8 9 10 11 12

# 1A # 4C homokaryon homokaryon 1, 4, 7, 10: MIH1-G1 + V2PDC1P → 506 bp 2, 5, 8, 11: MIH1-G4 + V3PDC1T → 401 bp 3, 6, 9, 12: MIH1-G1 + MIH1-I1 → 503 bp (wt)

Appendix 1.4. PCR verfification of Agcdc12∆mih1∆ (AgHPH27) homokaryons. (A) Verification of correct integration of the GEN3 cassette at the AgCDC12 locus and absence of the wt allele (B) Verification of the homokaryotic Agmih1∆ background. The Promega 1 kb DNA ladder was used as marker (lowest three bands: 750, 500 and 250 bp). AgHPH28 (Aghsl1∆::NAT1, Agmih1∆::GEN3) wild-type allele A

1 2 3 4 5 6 7 8 9 10 11 12

# 7 # 7B # 7F heterokaryon homokaryon homokaryon 1: HSL1-NG1 + V2PDC1P → 501 bp 2: HSL1-NG4 + V3PDC1T → 426 bp 3, 7: HSL1-NG4 + V3PDC1T → 426 bp 4, 8, 11: HSL1-I2 + HSL1NG4 → 264 bp (wt) 5, 9: MIH1-G1 + G2.1 → 508 bp 6, 10, 12: MIH1-G1 + I1 → 503 bp (wt)

AgHPH29 (Aghsl1∆::GEN3, Agmih1∆::NAT1) wild-type allele B

1 2 3 4 5 6 7 8

# 1 # 2 # 3 1, 3, 5, 7: HSL1-NG1.2 + G2.1 → 500 bp heterok. heterok. heterok. 2, 4, 6, 8: HSL1-NG4.2 + G3 → 536 bp (Aghsl1∆) (Aghsl1∆) (Aghsl1∆)

Appendix 1.5. Verfification of Aghsl1∆mih1∆ strains. (A) For the construction of AgHPH28, spores from heterokaryotic AgHPH23 (Agmih1∆::GEN3) were grown under selection and transformed with the Aghsl1∆::NAT1 cassette. The Agmih1∆ background was verified by one end only, since both ends were already verified in AgHPH23. (B) For AgHPH29, spores from homokaryotic AgHPH22 (Agmih1∆::NAT1) were used. Verification of the homokaryons including Agmih1∆ background is shown in Appendix 1.6. AgHPH29 (Aghsl1∆::GEN3, Agmih1∆::NAT1) wild-type allele A

1 2 3 4 5 6 7 8 9 10 11 12 13

# 1B # 1C # 2A homokaryon homokaryon homokaryon 1, 4, 7, 10: HSL1-G1.2 + G2.1 → 500 bp 2, 5, 8, 11: HSL1-G4.2 + G3 → 536 bp 3, 6, 9, 12, 13: HSL1-IA + HSL1-IB → 511 bp (wt)

AgHPH29 (Aghsl1∆::GEN3, Agmih1∆::NAT1) wild-type allele B

1 2 3 4 5 6 7 8 9 10 11 12 13

# 1B # 1C # 2A homokaryon homokaryon homokaryon 1, 4, 7, 10: MIH1-G1 + V2PDC1P → 506 bp 2, 5, 8, 11: MIH1-G4 + V3PDC1T → 401 bp 3, 6, 9, 12, 13: MIH1-G1 + MIH1-I1 → 503 bp (wt)

Appendix 1.6. PCR verfification of Aghsl1∆mih1∆ (AgHPH29) homokaryons. (A) Verification of correct integration of the GEN3 cassette at the AgHSL1 locus and absence of the wt allele. (B) Verification of the homokaryotic Agmih1∆ background. The Promega 1 kb DNA ladder was used as marker (lowest three bands: 750, 500 and 250 bp). AgHPH30 (Aghsl7∆::GEN3, Agmih1∆::NAT1) wild-type allele A

1 2 3 4 5 6 7 8

# 2 # 5 heterokaryon heterokaryon

1, 3, 5, 7: HSL7-G1.2 + G2.1 → 533 bp 2, 4, 6, 8: HSL7-G4.2 + G3 → 541 bp

AgHPH30 (Aghsl7∆::GEN3, Agmih1∆::NAT1) wild-type allele B

1 2 3 4 5 6 7 8 9 10

# 2A # 2B # 5A homokaryon homokaryon homokaryon

1, 3, 5, 7, 9: HSL7-G4.2 + G3 → 541 bp 2, 4, 6, 8, 10: HSL7-G4.2 + HSL7-I2.2 → 495 bp (wt)

Appendix 1.7. Verfification of Aghsl7∆mih1∆ strains. (A) Verification of heterokaryons. Unspecific bands are the result of low annealing temperature. The pattern of true transformants can clearly be distinguished from false positives or the reference strain. (B) Verfification of homokaryons. Agmih1∆ background. Spores from homokaryotic AgHPH22 (Agmih1∆::NAT1) were used for transformation. AgHPH31 (Agswe1∆::GEN3, Agmih1∆::NAT1) A

1, 3, 5: SWE1-G1 + G2.1 → 721 bp 2, 4, 6: SWE1-G4 + G3 → 824 bp

1 2 3 4 5 6

# 7 # 18 # 15 heterokaryon heterokaryon heterokaryon

AgHPH31 (Agswe1∆::GEN3, Agmih1∆::NAT1) B

1, 4, 7: MIH1-G1 + V2PDC1P → 506 bp 2, 5, 8: MIH1-G4 + V3PDC1T→ 401 bp 3, 6, 9: MIH1-G1 + MIH1-I1 → 503 bp (wt)

1 2 3 4 5 6 7 8 9

# 7 # 15 # 18 heterokaryon heterokaryon heterokaryon

AgHPH31 (Agswe1∆::GEN3, Agmih1∆::NAT1) wild-type allele C

1, 3: SWE1-G1 + G2.1 → 721 bp 2, 4, 7: SWE1-G1+ SWE1-I1 → 839 bp 5, 8: MIH1-G1 + MIH1-I1 → 503 bp (wt) 6: MIH1-G1 + V2PDC1P → 506 bp

1 2 3 4 5 6 7 8

# 15B # 7C Appendix 1.8. Verfification of Agswe1∆mih1∆ strains. (A) homokaryon homokaryon heterokaryons, (B) Agmih1∆ background, (C) homokaryons AgHPH17 (Agcdc11A∆::GEN3) AgHPH18 (Agcdc11B∆::GEN3) A

1, 3, 5, 7: CDC11A-G1 + G2.1 → 603 bp 2, 4, 6, 8: CDC11A-G4 + G3 → 542 bp 9, 11: CDC11B-G1 + G2.1 → 612 bp 10, 12: CDC11B-G4 + G3 → 636 bp

1 2 3 4 5 6 7 8 9 10 11 12

# 1 # 4 # 5 # 2 # 5 heterok. heterok. heterok. heterok. heterok.

AgHPH17 AgHPH18 AgHPH18 AgHPH17 (Agcdc11A∆) (Agcdc11B∆) (Agcdc11B∆) (Agcdc11A∆) B

1, 3, 5: CDC11B-G1 + G2.1 → 612 bp 2, 4, 6: CDC11B-G4 + G3 → 636 bp 7, 9, 11: CDC11A-G1 + G2.1 → 603 bp 8, 10, 12: CDC11A-G4 + G3 → 542 bp

1 2 3 4 5 6 7 8 9 10 11 12

# 1 # 5 # 2 # 2 # 5 #5 heterok. heterok. heterok. heterok. heterok. heterok.

AgHPH17 wild-type AgHPH18 wild-type (Agcdc11A∆::GEN3) allele (Agcdc11B∆::GEN3) allele C

1, 3: CDC11A-G4 + G3 → 542 bp 2, 4, 5: CDC11A-G4 + CDC11A-I2→ 485 bp (wt) 6, 8: CDC11B-G4 + G3 → 636 bp 7, 9, 10: CDC11B-G4 + CDC11B-I2→ 681 bp (wt)

1 2 3 4 5 6 7 8 9 10

# 1C # 5A? # 2A # 5C homokaryon homokaryon homokaryon homokaryon

Appendix 1.9. Verfification of Agcdc11A∆ and Agcdc11B∆ strains. (A) heterokaryons, (B) CDC11A verification oligos G1 and G4 were tested for unspecific binding to the AgCDC11B promotor and terminator region in Agcdc11B∆ strains and vice versa. No crossreaction was detected. (C) Verification of homokaryons. AgHPH36 (Agcdc28Y18F-GEN3) wild-type allele A

1 2 3 4 5 6 7 8 9 10 11 12 13 14

# 12A # 23A # 12A false # 23A homok. homok. homok. homok.

1, 2, 3, 6, 9, 12: CDC28-Y18F-IA + CDC28-IB → 565 bp (Y18F-IA binds mutated seq.) 4, 7, 10, 13: CDC28-G4.2 + G3 → 704 bp 5, 8, 11, 14: CDC28-IA-Y18 + CDC28-IB → 564 bp (IA-Y18 binds wt seq.)

wt wt AgHPH36 (Agcdc28Y18F-GEN3) allele AgHPH36 (Agcdc28Y18F-GEN3) allele B

#8A #12A #22A #23A #8A #12A #22A #23A false true false true false true false true CDC28-G1.2 + CDC28-IB → 732 bp KpnI AgHPH36 → 472, 172, 88 bp uncleaved KpnI reference → 644, 88 bp

Appendix 1.10. Verification of Agcdc28Y18F strains. (A) Verification by PCR. CDC28- Y18F-IA binds specifically to the mutated sequence (incl. Phe18), but binding is weak. Bands that were seen by eye on a UV table but were to weak for printing using the gel imager are painted in green. CDC28-G4.2 + G3 was used to confirm correct integration of the GEN3 module. CDC28-IA-Y18 was designed to bind to the wild-type sequence, only (incl. Tyr 18), but results in unspecific, weaker bands in the mutant strains. The pattern is clearly distinguishable between true mutants and false positives or the wild-type allele. A band of correct size (564 pb) is only observed for false positives or the wild-type allele, not for true positives. The lowest band in true positives corresponds to the second lowest (false) band in wild type. (B) A 732 bp fragment including Tyr 18 / Phe 18 was amplified and digested with KpnI, which cuts the mutant fragment three times and the wild-type fragment twice. Additional bands are likely to be the result of the 1.5 kb unspecific PCR product. SacI 6xGA

quence . se A.g G-S1 AER013W 'lacZ AER014W GFP AgHHF1 AER015C FW GFP 5000 BglII ScURA3-T ScADH1-T ampR 'AER016C AgTEF2-P 4000 pGUC 1000 pAG-H4-GFP-KanMX6

ScPDC1-P R 5023 bps 10840 bps kan

3000 2000 AgTEF2-T NAT1 R amp RW

ScPDC1-T ScURA3 G-CS2

HindIII Upstream homology ampR URA3 ampR I n t e g partial r a

t i AgADE2 o n UP

pHPH001-2R C pAG4401

t 6153 bps t 9184 bps

e ABL158C RP

ScADH1-T AgSEP7 AER013W' GFP (ABL159W) AgHHF1 )S2 EcoRI S7-G-S1 G(C Downstream A S7- .g. homology A se .g. que seque nce nce del

Appendix 2.1. pAG-H4-GFP-KanMX6 was constructed by C. Alberti-Segui, but no map or sequence was provided. We reconstructed the map based on information gathered from Alberti-Segui et al., 2001 and Alberti-Segui, Thesis, 2001. pAG4401 is part of the pAG collection created by C. Mohr (C. Mohr, Thesis, 1995; Dietrich et al., 2004). The map is shown to illustrate the construction of pAGHPH002 and pAGHPH003 (see Appendix 2.2). ScURA3 ScURA3 ampR ampR

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pAGHPH003 RP pAGHPH002 ABL158C RP (pSep7-GUC) ABL158C (pSep7-GUG) 11492 bps 11862 bps

A AgSEP7 AgSEP7 A . g (ABL159W) . (ABL159W) . S7-G-G4 g S7-G-CG4

s . s e S7-G-G1 q e S7-G-G1 u G3 q u V3PDC1T e e n GG2 n c R c GG2 e kan e NAT1 ScTEF2-T GFP GFP ScPDC1-T

6xGA 6xGA ScPDC1-P ScURA3-T ScTEF2-P ScURA3-T

EcoRI EcoRI

ampR ampR A A . g .g 'ABL158C . . s 'ABL158C s e e q q u u e e

n n

c c

e e pUCHPH002F pUCHPH003F S7-G-CG4 S7-G-G4 'AgSEP7 7118 bps 6748 bps EcoRI 'AgSEP7 (ABL159W) E (ABL159W) c G3 oRI V3PDC1T GG2 ScTEF2-T GG2 6xGA kanR GFP NAT1 ScPDC1-T 6xGA GFP

ScURA3-T ScTEF2-P ScURA3-T ScPDC1-P

Appendix 2.2. A AEL148W' . AEL148W' g. s e q u e ScURA3 n c ScURA3 e AgSWE A (AEL149C) . g

pAGHPH004 u pAGHPH005 e

(pAGSWE1) n (pAGSWE1-MYC) c 7954 bps AgSWE e 10447 bps 13myc (AEL149C) ampR

ampR ScTEF2-T kanR

ScTEF2-P

AEL148W' A AEL148W' A .g ScaI .g . . s s e e q q u u ScURA3 e ScURA3 e n n c c e AgSEP7 AgSEP7 e (ABL159W) (ABL159W) MscI pAGHPH006 pAGSWE1-HA-NAT1 (pAGSWE1-GUC) 6xGA 10263 bps 9587 bps 6HA ampR GFP ampR ScPDC1-P ScURA3-T ScaI NAT1 ScaI NAT1

ScPDC1-T ScPDC1-P

ScPDC1-T ScaI

Appendix 2.3. pAGSWE1-HA-NAT1 was created by A. Gladfelter. PstI PstI

ScURA3 ScURA3 ampR ampR

KpnI KpnI pRS416CDC28GEN3 pAGHPH007 XhoI XhoI (pAGCDC28Y18F) EcoRI EcoRI 8244 bps 8244 bps ScTEF2-T ScTEF2-T G3 G3 AgCDC28 AgCDC28 'ADR059C 'ADR059C R (ADR058C) R (ADR058C) kan CDC28-G1.2 kan CDC28-G1.2 CDC28-IA-Y18 CDC28-Y18F-IA BamHI BamHI CDC28-IB CDC28-IB Y18 Y18F alt. codons XhoI KpnI ScTEF2-P ScTEF2-P KpnI KpnI

ScTEF2-T AEL148W' ScTEF2-T AEL148W' ScURA3 ScURA3 R kanR kan

ScTEF2-P ScTEF2-P pAGHPH008 pAGHPH009 (pAGHIS-SWE1) ScHIS3-P (pAGHIS-SWE1-HA) ScHIS3-P 9777 bps 9890 bps ampR ampR AgSWE AgSWE (AEL149C) (AEL149C) rt e s t n r -I se g n -I 6HA A Ag ScPDC1P'

MscI MscI XbaI

Appendix 2.4. pRS416CDC228GEN3 was constructed by A. Gladfelter and is shown here to illustrate the methods used to distinguish mutant from reference strains (see Appendix 1.7). 10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc28 MS-DLTNYKRLEKVGEGTYGVVYKAVDLR--HGQRIVALKKIRLESEDEGVPSTAIREIS 57 ScCdc28 MSGELANYKRLEKVGEGTYGVVYKALDLRPGQGQRVVALKKIRLESEDEGVPSTAIREIS 60

70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc28 LLKELKDDNIVRLYDIVHSDAHKLYLVFEFLELDLKRYMESVPKDQPLGDKIIKKFMMQL 117 ScCdc28 LLKELKDDNIVRLYDIVHSDAHKLYLVFEFLDLDLKRYMEGIPKDQPLGADIVKKFMMQL 120

130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc28 CKGIAYCHAHRIIHRDLKPQNLLINRNGNLKLGDFGLARAFGVPLRAYTHEIVTLWYRAP 177 ScCdc28 CKGIAYCHSHRILHRDLKPQNLLINKDGNLKLGDFGLARAFGVPLRAYTHEIVTLWYRAP 180

190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc28 EVLLGGKQYSTGVDVWSIGCIFAEMCNRKPLFSGDSEIDQIFKIFRLLGTPNESVWPDIV 237 ScCdc28 EVLLGGKQYSTGVDTWSIGCIFAEMCNRKPIFSGDSEIDQIFKIFRVLGTPNEAIWPDIV 240

250 260 270 280 290 ....|....|....|....|....|....|....|....|....|....|....|... AgCdc28 YLPDFKPTFPKWQRRDLAQVVPSLNEHGLDLLDKLVTYDPIHRISAKRAVTHPYFKDE 295 ScCdc28 YLPDFKPSFPQWRRKDLSQVVPSLDPRGIDLLDKLLAYDPINRISARRAAIHPYFQES 298

Appendix 3.1. Alignment of AgCdc28p and ScCdc28p (1/1) 10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| AgSwe1p MTLRHDSMAALRESPFLNRKHTL-SPALRFNCMVHEEEELSEADLESEGDSDRTQPATPS 59 ScSwe1p ------MSSLDEDE--EDFEMLDTENLQF----MGKKMFGKQAGEDESDDFAIGGSTPT 47

70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| AgSwe1p NNLRFYSSRQAAGLCRSVGTLNLSLENASRKLAPLTTSRDLPVRVREEDNGGLEFLSGTA 119 ScSwe1p NKLKFYPYSNNK-LTRSTGTLNLSLSNTA---LS-EANSKFLGKIEEEEEEEEE------96

130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....| AgSwe1p MCQGDGDKEFHRGDNLWRPFPPRHDKNLKRSASKCEMKPPLISDRSQSRDNDSVEDENAR 179 ScSwe1p ---GKDEESVDSRIKRWSPF---HENE------SVTTP-ITKRSAEKTNSPI-SLKQW 140

190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....| AgSwe1p NMAYSPLGNKKKMRKFPECQLFKDVKPDQSAFQQKGLMSKM-R-SNLLPQKLIIPDTPVK 237 ScSwe1p NQRWFPK-NDARTENTSSSSSYSVAKPNQSAFTSSGLVSKMSMDTSLYPAKLRIPETPVK 199

250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....| AgSwe1p KSP------HSSI-MESPMD-CSV-YGSSSVPHDTQPFKNFPLFK--PG--TPP 278 ScSwe1p KSPLVEGRDHKHVHLSSSKNASSSLSVSPLNFVEDNNLQEDLLFSDSPSSKALPSIHVPT 259

310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....| AgSwe1p IESSP----TY--Q--HRAQR------LD-SKNDM-PA--QRMKR-RSKVI-KNTD 314 ScSwe1p IDSSPLSEAKYHAHDRHNNQTNILSPTNSLVTNSSPQTLHSNKFKKIKRARNSVILKNRE 319

370 380 390 400 410 420 ....|....|....|....|....|....|....|....|....|....|....|....| AgSwe1p LSNMLQQFTDDLFG------S--G-D-EEPVFNSSPLR- 342 ScSwe1p LTNSLQQFKDDLYGTDENFPPPIIISSHHSTRKNPQPYQFRGRYDNDTDEEISTPTRRKS 379

430 440 450 460 470 480 ....|....|....|....|....|....|....|....|....|....|....|....| AgSwe1p ----TPRK-RSPQPDRLRPP-VTTTRQQGKRRL--VGTSHA-S--Q---SARAR-NPDEH 387 ScSwe1p IIGATSQTHRESRPLSLSSAIVTNTTSAETHSISSTDSSPLNSKRRLISSNKLSANPDSH 439

490 500 510 520 530 540 ....|....|....|....|....|....|....|....|....|....|....|....| AgSwe1p LGANFSNVTLLGRGQFSTVYQVTFPETSAKYAVKSMAPKKHYSRSRIIQEIQLLSEISQE 447 ScSwe1p LFEKFTNVHSIGKGQFSTVYQVTFAQTNKKYAIKAIKPNKYNSLKRILLEIKILNEVTNQ 499

550 560 570 580 590 600 ....|....|....|....|....|....|....|....|....|....|....|....| AgSwe1p -TSDAEGREYVVQFISSWEYQGTYYAMTELCENGNLDQFLQEQLVARSKRLEDWRIWKII 506 ScSwe1p ITMDQEGKEYIIDYISSWKFQNSYYIMTELCENGNLDGFLQEQVIAKKKRLEDWRIWKII 559

610 620 630 640 650 660 ....|....|....|....|....|....|....|....|....|....|....|....| AgSwe1p VEVCLGLRFLHETCSIVHLDLKPANIMITFEGNLKLGDFGMATKLPLTDKAFENEGDREY 566 ScSwe1p VELSLALRFIHDSCHIVHLDLKPANVMITFEGNLKLGDFGMATHLPLEDKSFENEGDREY 619

670 680 690 700 710 720 ....|....|....|....|....|....|....|....|....|....|....|....| AgSwe1p IAPEIISDGIYDFRADIFSLGLMIVEIAANVVLPDNGNAWHKLRSGDLSDAGRLSSTEI- 625 ScSwe1p IAPEIISDCTYDYKADIFSLGLMIVEIAANVVLPDNGNAWHKLRSGDLSDAGRLSSTDIH 679

Appendix 3.2. Alignment of AgSwe1p and ScSwe1p (1/2) 730 740 750 760 770 780 ....|....|....|....|....|....|....|....|....|....|....|....| AgSwe1p ----Y------TTSI--F------S----STDINSTNITEVSRPCSYNAGS 654 ScSwe1p SESLFSDITKVDTNDLFDFERDNISGNSNNAGTSTVHNNSNINNPNMNNGNDNNNVNTAA 739

790 800 810 820 830 840 ....|....|....|....|....|....|....|....|....|....|....|....| AgSwe1p G--R--I-KH--IPAWVPRFLIDNDSLEKLVIWMIEPDYRKRPTASNLLQAEECQYVELT 707 ScSwe1p TKNRLILHKSSKIPAWVPKFLIDGESLERIVRWMIEPNYERRPTANQILQTEECLYVEMT 799

850 860 ....|....|....|....| AgSwe1p RKAGAIIQEDDFGPKPEFFS 727 ScSwe1p RNAGAIIQEDDFGPKPKFFI 819

Appendix 3.2. Alignment of AgSwe1p and ScSwe1p (2/2) 10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| AgMih1p ------MQEAGD-SK-G-RRSSFHGGSSSF---LK--NL-NSLL-K- 30 ScMih1p MNNIFHGTEDECANEDVLSFQKISLKSPFGKKKNIFRNVQTFFKSKSKHSNVDDDLINKE 60

70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| AgMih1p ---LKKSP------GRPAEGAEP-----ANEPHLGDNAKERDR-GLCTR-PPGPVSRCST 74 ScMih1p NLAFDKSPLLTNHRSKEIDGPSPNIKQLGHRDELDENENENDDIVLSMHFASQTLQSPTR 120

130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....| AgMih1p GTVYR--SPSHRRQ--G----SSASAKR-EFKPSHATAVQHASHCCNCMLRGMQPPADSP 125 ScMih1p NSSRRSLTNNRDNDLLSRIKYPGSPQRSSSFSRSRSLSRKPSMNSSSNSSRRVQRQDGKI 180

190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....| AgMih1p AERGVTRGEAF------A-N--PVCSRGLESKSVPPYVYDSRLPASAIPWHTKDSSTDQ- 175 ScMih1p PRSSRKSSQKFSNITQNTLNFTSASSSPLAPNSVGVKCFESCLAKTQIPYYYDDRNSNDF 240

250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....| AgMih1p LPRISVDVLAAILDGKF-SSHYSEVYIIDCRFEYEFQAGHIKNAINVSSRRELEAEFIQK 234 ScMih1p FPRISPETLKNILQNNMCESFYNSCRIIDCRFEYEYTGGHIINSVNIHSRDELEYEFIHK 300

310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....| AgMih1p RIQR-CSADPGRPPLLVFHCEYSSYRGPIIAAHLRNYDRILNHGQYPRLHYPDIVVLQGG 293 ScMih1p VLHSDTSNNNTLPTLLIIHCEFSSHRGPSLASHLRNCDRIINQDHYPKLFYPDILILDGG 360

370 380 390 400 410 420 ....|....|....|....|....|....|....|....|....|....|....|....| AgMih1p FKSFIEAFPGFC-QGHYVGM---DSYVNHELELARFKRDS-----K--TI--LTRQNSQH 340 ScMih1p YKAVFDNFPELCYPRQYVGMNSQENLLNCEQEMDKFRRESKRFATKNNSFRKLASPSNPN 420

430 440 450 460 470 480 ....|....|....|....|....|....|....|....|....|....|....|....| AgMih1p IFQEQGQEGGHGLAGRA--YQPDPVATLLPTRRPSFSFDAPPQSPSLVIGGNSGSSA-CS 397 ScMih1p FFYRDSHQSSTTMASSALSFRFEPPPKLSLNHRR-VS-SGSSLNSSESTGDENFFPILSK 478

490 500 510 520 530 540 ....|....|....|....|....|....|....|....|....|....|....|....| AgMih1p CSSSTRSVL-TGKMLLMHDL-TT------DVKACDKDDQDDQFSFDLGDELAITV 444 ScMih1p SSMSSNSNLSTSHMLLMDGLDTPSYFSFEDERGNHQQVSGDEEQDGDFTF-----VGSDR 533

550 560 ....|....|....|....|.... AgMih1p EDLFSSPVGRRLFPEIVKEEDNDS 468 ScMih1p EDL-PRPARRSLFPS-L-ETEDKK 554

Appendix 3.3. Alignment of AgMih1p and ScMih1p (1/1) 10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc5p MS-APLQIVNEKQLNTRS---N--ANVHTP--V-KQHHAKRADLERGHHDNNRQPPQKKK 51 ScCdc5p MSLGPLKAINDKQLNTRSKLVHTPIKGNTADLVGKENHFKQTKRLDPNNDHHHQPAQKKK 60

70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc5p KEKLSALCKTPPSLIKTKGRDYHRGMFLGEGGFARCFQMKDDSGKVFAAKTVAKISIKSE 111 ScCdc5p REKLSALCKTPPSLIKTRGKDYHRGHFLGEGGFARCFQIKDDSGEIFAAKTVAKASIKSE 120

130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc5p KTRKKLLSEIQIHKSMKHPNIVQFTDCFEDDTNVYILLEICPNGSLMDLLKQRKQLTEPE 171 ScCdc5p KTRKKLLSEIQIHKSMSHPNIVQFIDCFEDDSNVYILLEICPNGSLMELLKRRKVLTEPE 180

190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc5p VRFFTTQIVGAIKYMHSRRIIHRDLKLGNIFFDKHFNLKIGDFGLAAVLANDRERKYTIC 231 ScCdc5p VRFFTTQICGAIKYMHSRRVIHRDLKLGNIFFDSNYNLKIGDFGLAAVLANESERKYTIC 240

250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc5p GTPNYIAPEVLTGKHTGHSFEVDIWSIGVMIYALLIGKPPFQAKEVNTIYERIKVCDFSF 291 ScCdc5p GTPNYIAPEVLMGKHSGHSFEVDIWSLGVMLYALLIGKPPFQARDVNTIYERIKCRDFSF 300

310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc5p PKDKPISSEAKVLIKDILSLDPLERPSLAEIMEYVWFRNVFPARINGDILNFVPEFHDLD 351 ScCdc5p PRDKPISDEGKILIRDILSLDPIERPSLTEIMDYVWFRGTFPPSIPSTVMSEAPNFEDIP 360

370 380 390 400 410 420 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc5p LQESLINFKNCMAKCGLMNPSAAAAAVAAKEYDQRLPSYSKNSNNNNNQINQNNEQLSEA 411 ScCdc5p EEQSLVNFKDCMEK-SLLLE-----SMSSDKIQRQKRDYI--SSIKSS-IDKL-EEYHQ- 409

430 440 450 460 470 480 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc5p KKSVLPQSLSPGGTRSS-PGNCYVETQRKLNDLAREARIRRAQQSMLKQSLVASSTNFIK 470 ScCdc5p NRPFLPHSLSPGGTKQKYKEVVDIEAQRRLNDLAREARIRRAQQAVLRKELIATSTNVIK 469

490 500 510 520 530 540 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc5p SEISLRILASECHMTLNGLLEAEAQKRMGGLPRSRLPEIQHPIVVTKWVDYSNKHGFAYQ 530 ScCdc5p SEISLRILASECHLTLNGIVEAEAQYKMGGLPKSRLPKIKHPMIVTKWVDYSNKHGFSYQ 529

550 560 570 580 590 600 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc5p LSTDDIGVLFNNGTTVLNLADAEEFWYISYDDREGWVANHYSLAEKPKELNRHLEVVDFF 590 ScCdc5p LSTEDIGVLFNNGTTVLRLADAEEFWYISYDDREGWVASHYLLSEKPRELSRHLEVVDFF 589

610 620 630 640 650 660 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc5p SNYMNSNLSRISTFVRESYHKDDVFLRRFTRYKQFVMFELSDGTFQFNFKDHHKFAISQS 650 ScCdc5p AKYMKANLSRVSTFGREEYHKDDVFLRRYTRYKPFVMFELSDGTFQFNFKDHHKMAISDG 649

670 680 690 700 710 ....|....|....|....|....|....|....|....|....|....|....|... AgCdc5p GKLTTYISPDRQSFTFPTVEILKAEKIPGHPELGFMEKFAMIKEGLKQKSAIVSVAQQ 708 ScCdc5p GKLVTYISPSHESTTYPLVEVLKYGEIPGYPESNFREKLTLIKEGLKQKSTIVTV--D 705

Appendix 3.4. Alignment of AgCdc5p and ScCdc5p (1/1) 10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p ------MT-----A-GHR-DERP-DR-EGGSER-DEHLNRVVQSVSDATKRLSQIS 40 ScHsl1p MTGHVSKTSHVPKGRPSSLAKKAAKRAMAKVNSNPKRASGHLERVVQSVNDATKRLSQPD 60

70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p NSSTAHTKTGKRKNRDTVGPWKLGKTLGKGSSGRVRLAKNMQSGKLAAIKIVP-KR---- 95 ScHsl1p STVSVATKSSKRKSRDTVGPWKLGKTLGKGSSGRVRLAKNMETGQLAAIKIVPKKKAFVH 120

130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p ------N-V-RHNQKQVTALPYGIEREIIIMKLITHPNIMALY 130 ScHsl1p CSNNGTVPNSYSSSMVTSNVSSPSIASREHSNHSQTNPYGIEREIVIMKLISHTNVMALF 180

190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p EVWENKSELYLVLEYVEGGELFDYLIARGKLPEQEAIHYFKQIVQGVSYCHNFNICHRDL 190 ScHsl1p EVWENKSELYLVLEYVDGGELFDYLVSKGKLPEREAIHYFKQIVEGVSYCHSFNICHRDL 240

250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p KPENLLLDKKNKTVKIADFGMAALETTNRLLETSCGSPHYASPEIVMGQKYHGSPSDVWS 250 ScHsl1p KPENLLLDKKNRRIKIADFGMAALELPNKLLKTSCGSPHYASPEIVMGRPYHGGPSDVWS 300

310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p CGIILFALLTGHLPFNDDNVRKLLLKVQHGRYQMPSNVSKEAKDLISKILVVDPEKRITV 310 ScHsl1p CGIVLFALLTGHLPFNDDNIKKLLLKVQSGKYQMPSNLSSEARDLISKILVIDPEKRITT 360

370 380 390 400 410 420 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p DKILEHPLLVKY------DNPGKGGLACPLEKPEER-PKVLDIHSIDDIDET 355 ScHsl1p QEILKHPLIKKYDDLPVNKVLRKMRKDNMARGKSNSDLHLLNNVSPSIVTLHSKGEIDES 420

430 440 450 460 470 480 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p ILSNLQILWHGAPKEYLVDKLLQSGFTEEKLFYALLLRYQQRQSV------E 401 ScHsl1p ILRSLQILWHGVSRELITAKLLQKPMSEEKLFYSLLLQYKQRHSISLSSSSENKKSATES 480

490 500 510 520 530 540 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p PTSVPSNM--ASTNGSGAFA------RAS--NN-D------QSVINAPKV 434 ScHsl1p SVNEPRIEYASKTANNTGLRSENNDVKTLHSLEIHSEDTSTVNQNNAITGVNTEINAPVL 540

550 560 570 580 590 600 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p TQKSQFSINSL--LVSPKKTRE------G---YVASSSRVFKH------SVSRRSLHVSP 477 ScHsl1p AQKSQFSINTLSQPESDKAEAEAVTLPPAIPIFNASSSRIFRNSYTSISSRSRRSLRLSN 600

610 620 630 640 650 660 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p S--PM---KSSTSCR------S-L---KSS-S-SKR-L-QSPNPKAITQPV---KVTSPQ 515 ScHsl1p SRLSLSASTSRETVHDNEMPLPQLPKSPSRYSLSRRAIHASPSTKSIHKSLSRKNIAATV 660

670 680 690 700 710 720 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p PRRRTLHNSASKRSLYSLTSISKRSLNLNEFLQEANEVKTPKLPSMPLQDQPRSEAPKTP 575 ScHsl1p AARRTLQNSASKRSLYSLQSISKRSLNLNDLLVFDDPLPSKK--PAS-ENVNKSEPHSLE 717

730 740 750 760 770 780 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p TKTTYGADSHGGEKPTFISVLHDCPTVDSEGMVFKSSSGTPMIAANAPIIQPAF-IFGVG 634 ScHsl1p SDSDF--EILC-DQILFGNAL-D-RILEEEEDNEKERDTQRQRQNDTKSSADTFTISGVS 772

790 800 810 820 830 840 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p AFPGTKQPS-SNSLSKPSLNSQSSPKQAQEATAN---SMRETIRDRQTLVVEKRVPSKTE 690 ScHsl1p TNKENEGPEYPTKIEKNQFNMSYKPSENMSGLSSFPIFEKENTLSSSYLEEQKPKRAALS 832

Appendix 3.5. Alignment of AgHsl1p and ScHsl1p (1/2) 850 860 870 880 890 900 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p SVPPAFPRVRPIMERKHGSDLTLVRDTRSVAKEKRSSLSLDPRRTAPEPPRGIETLLRRY 750 ScHsl1p DITNSF-N-K--M-NKQEG----MRIEKKIQRE-Q----LQKKNDRP-SP------867

910 920 930 940 950 960 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p SLKNSIKSRSSLKKKRDSGAWSITDKTIFQDLGQVPEDEEQEKDSEEKGNDTFISTTETS 810 ScHsl1p -LK-PIQ-HQELRVNSLPN--D-QGKPSL-SL-D-PRRNIS-QPVNSKV-ESLLQGLKFK 916

970 980 990 1000 1010 1020 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p DTPSSKFKVTTSTPIKKDDATAISAENAAVGTEQSGSEDEATNFDSNAHSYPISTDLKTE 870 ScHsl1p KEPASHWTHERGSLFMSEH---VEDEKPVKASDV--SI-E-SSYVPLT-T--VATSSRDP 966

1030 1040 1050 1060 1070 1080 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p DDLTPIKPQGQTDLLRMPSSFMNSSNTFANLNNFIS-N-IDKEYSPENSDSGRIEKDEPA 928 ScHsl1p SVLAESSTI-QKPMLSLPSSFLNTSMTFKNLSQILADDGDDKHLSVPQNQSRSVAMSHPL 1025

1090 1100 1110 1120 1130 1140 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p GKAQPEQITVKKRQRTLAPGGYLSPSIPRMESENSLNQELGSRISDLSDLSFANDMPTYT 988 ScHsl1p -RK---Q-SA-KI--SLTPRSNLNANLSVKRNQGSPGSYLSNDLDGISDMTFAMEIPTNT 1077

1150 1160 1170 1180 1190 1200 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p NTAHAVS-I--SPGTRNVCSFSPLEPRSPQDVSNNGKMRSNLHIPLRQLSEDMCDKTI-T 1044 ScHsl1p FTAQAIQLMNNDTDNNKINTSPKASSFTKEKVIKSAAYISKEKEPDNSDTNYIPDYTIPN 1137

1210 1220 1230 1240 1250 1260 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p TQEGESVNIFEDAPEEEGSL-TTST-SGSVPNIHQKAISIDTMNTSCALVPVSHVRTSIH 1102 ScHsl1p TYDEKAINIFEDAPSDEGSLNTSSSESDSRASVHRKAVSIDTMATTNVLTPATNVRVSLY 1197

1270 1280 1290 1300 1310 1320 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p MNGDSSVLDWSTSDGILSKAR---PLPTSNHVRKK----SGNRLSNNLTLSKSMMSMFKN 1155 ScHsl1p WNNNSSGIPRETTEEILSKLRLSPENPSNTHMQKRFSSTRGSRDSNALGISQSLQSMFKD 1257

1330 1340 1350 1360 1370 1380 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p L--DQINVTDIQDPMPVSDLLSKGVDPIEPIEPLKNDQVTLKAESIGAESDSINMDLADR 1213 ScHsl1p LEEDQDGHTSQADILESSMSYSKRRPSEESVNP--KQRVTMLFDEEEEESKKVGGGKIKE 1315

1390 1400 1410 1420 1430 1440 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p KHVTIFHDNVSDKNFKSTRMENSDKGSLGSGAPQATHLFHTGNKDMKSHETLLSPITETP 1273 ScHsl1p EH-TKLDNKISE---ESSQLVLPVVEK--KENANNTENNY---SKIPK-PSTIKVTKDTA 1365

1450 1460 1470 1480 1490 1500 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p AAALSATSLTRKESISSDGKSVKDETKKSGKGNWFMRLIMSFTKPKK-T-VIQEHMTVLP 1331 ScHsl1p MESNTQTH-TKKPILKSVQNVEVEEAPSSDKKNWFVKLFQNFSSHNNATKASKNHVTNIS 1424

1510 1520 1530 1540 1550 1560 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl1p FDDVNIITLQQFSYQGVEYEIRQMERRKDKQKVEYDCRLLDGDFKFKINIYGENSAATKV 1391 ScHsl1p FDDAHMLTLNEFNKNSIDYQLKNLDHKFGRKVVEYDCKFVKGNFKFKIKITSTPNASSVI 1484

1570 1580 1590 ....|....|....|....|....|....|....|... AgHsl1p SVKQKGR----SDELAFTKLNTDIEALIKAKENAANVK 1425 ScHsl1p TVKKRSKHSNTSSNKAFEKFNDDVERVI--R--NAGRS 1518

Appendix 3.5. Alignment of AgHsl1p and ScHsl1p (2/2) 10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl7p MKSNVFVGI------KG---HGVLE-GAAYREKYDYVLEGVTNGRYRD---AV-- 40 ScHsl7p MHSNVFVGVKPGFNHKQHSKKSRFLENVSSHSPELPSNYDYVLLPITTPRYKEIVGQVFK 60

70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl7p ---RAAAAA--GVAVSPPELAEVGGGP------GGGAGRLGLAAPWLELESAEPAIGE 87 ScHsl7p DFQRQSIQNWKPLQIPEPQLQDICIPPFNVKKLDNDDTPSYIGLLSSWLELESRDPNVRD 120

130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl7p VSLRVLEHEYEYARAEGVKQLIVAPPRELGRLNLYAQRLGRLWE-RAG-RGPPL-VSVSL 144 ScHsl7p LGLKVLLNECKYARFVGINKLILAPPRDLSNLQLYGQMIYRLLQNRIVFAAPALTISISL 180

190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl7p PLFEAGDPLSTWELWNTVRRLCRYHPNLTATLAVPRGRTPGHVLRRWLAEPVSCLLVSSS 204 ScHsl7p PLYEDSDPLATWELWNTVRKQCEYHPSLTISLALPRTRTPSYVLNRWLAEPVSCLLVSSS 240

250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl7p ILVTNQYNYPVLHKHNQELIGLFQRLNGRAESVLGELTIVLHGIEKHAERVRGGEPIYLE 264 ScHsl7p IFASNQYDYPVLHKFNQNLILKFQKVNGDSQILGNELCVILHGMEKYANNVKGGESAYLE 300

310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl7p YINYLLKKGDRAL-LQAPGE---DGAPRVMQPLQPHAVDLSSEVYQIFEQDKTKYDLYAK 320 ScHsl7p YINYLLKKGDKVLNSNSNHQFLLQEDSRIMPPLKPHSDNLLNSTYLTFEKDLVKYDLYES 360

370 380 390 400 410 420 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl7p AITAALRSIRSTMNKMWLHDDLNIIVVGAGRGGLVDRAYTCLRQLGI-SRFKLVALEKNP 379 ScHsl7p AILEALQDL-APRASAK-R-PLVILVAGAGRGPLVDRTFKIISMLFMDSKVSIIAIEKNP 417

430 440 450 460 470 480 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl7p QAVIHLQKKNIEKWGNSVDIVSANMREW------SSKVKFDLCISELLGSFGCNELAPEC 433 ScHsl7p QAYLYLQKRNFDCWDNRVKLIKEDMTKWQINEPSEKRIQIDLCISELLGSFGCNELSPEC 477

490 500 510 520 530 540 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl7p LEAFEKTNCTDRTIFIPQSYTSYVAPVSAPLLYQMLRNKEDNALESPWVVRNVPSCLLST 493 ScHsl7p LWSIEKYHSHNDTIFIPRSYSSYIAPISSPLFYQKL-SQTNRSLEAPWIVHRVPYCILSS 536

550 560 570 580 590 600 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl7p KVYELWSFKHP------GGTSN-T---ARSTVTNMKIKHKGEVHGLLGFFTAEIYGDIR 542 ScHsl7p RVNEVWRFEHPMAQKDTVQDEDDFTVEFSQSSLNEFKIKHRGEIHGFIGFFSANLYNNIF 596

610 620 630 640 650 660 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl7p LSILPDDCKIKLRGSAPDSDGRRKSVDLDSKLGHTPNMSSWSPIFFPLLYPMFVGDDTEL 602 ScHsl7p LSTLPNDSTVRLKFSEETLMNTRREENLIKKCDHTPNMTSWSPIIFPLKQPISFIDDSEL 656

670 680 690 700 710 720 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl7p ELTMLRNRC--MRGVWYEWSLSSYVYNAISQDRKPGSFLKGTQQLLNSKPEPNFKDTFTR 660 ScHsl7p SVLMSRIHSDTEQKVWYEWSLESFIY------LM------LSN-YT- 689

Appendix 3.6. Alignment of AgHsl7p and ScHsl7p (1/2) 730 740 750 760 770 780 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl7p SKKHDGLNSVMLDLQNTATILNAGDLGEPREAQYDGNIEENSASTELLKRTFVGKSE--F 718 ScHsl7p ----SAVTAASMTIPRSIVTDDTKTLAHNR--HYSATTNQKLDNQIDLDQDIENEEEQGF 743

790 800 810 820 830 840 ....|....|....|....|....|....|....|....|....|....|....|....| AgHsl7p ISPELTGWQSVHDVHELGQT-M-T--EKPNKGDYN------NSEYEDKDYSNYE--E 763 ScHsl7p LSNLETGWQSVQDIHGLSETAKPDHLDSINKPMFDLKSTKALEPSNELPRHEDLEEDVPE 803

850 860 ....|....|....|....|.... AgHsl7p YHVRVRTNTTELHNIGGHAYMIKL 787 ScHsl7p VHVRVKTSVSTLHNVCGRAFSLPL 827

Appendix 3.6. Alignment of AgHsl7p and ScHsl7p (2/2) 10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc3p MLPTGSQEDSANTNSATTAGGVTTSTGGALM--SNGTASAKDTSSDMNVKEEEMGLDLPD 58 ScCdc3p -MSLKEEQVSIKQDPEQEERQHDQFNDVQIKQESQDHDGVDSQYTNGTQNDDSERFEAAE 59

70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc3p DKENVATALG-GEL-A-A--GQVLPDQPDLRIIHRKISGYVGFANLPKQWHRKSIRRGFN 113 ScCdc3p SDVKVEPGLGMGITSSQSEKGQVLPDQPEIKFIRRQINGYVGFANLPKQWHRRSIKNGFS 119

130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc3p LNLLCVGAKGLGKSTLINTLFNK-ELYTA--KDDTPEQFNALKLEDGE-DGDESRAKDGE 169 ScCdc3p FNLLCVGPDGIGKTTLMKTLFNNDDIEANLVKDYEEELANDQEEEEGQGEGHENQSQEQR 179

190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc3p NKVKIETVTTEIEENGVVLKLTVVDTPGFGDAIDNTD-SWKPIVDEMNSRFDQYLDAENK 228 ScCdc3p HKVKIKSYESVIEENGVKLNLNVIDTEGFGDFLNNDQKSWDPIIKEIDSRFDQYLDAENK 239

250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc3p INRTTIDDNRIHACLYFIEPTGHCLKPLDLEFCRQVHDKCNLIPVIAKSDILTDEEIEHF 288 ScCdc3p INRHSINDKRIHACLYFIEPTGHYLKPLDLKFMQSVYEKCNLIPVIAKSDILTDEEILSF 299

310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc3p KWTIKKQLDDAKVHLFQPPQYLLDDEETQRATRQLFSKVPFAVVGSTHVGGHLRREGQFR 348 ScCdc3p KKTIMNQLIQSNIELFKPPIYSNDDAENSHLSERLFSSLPYAVIGSNDIVENY-SGNQVR 358

370 380 390 400 410 420 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc3p GRSYPWGIIEVDNEKHSDFVYLRDLLIRQYLEELRERTNNELYEKYRSEKLIRMGIKQDN 408 ScCdc3p GRSYPWGVIEVDNDNHSDFNLLKNLLIKQFMEELKERTSKILYENYRSSKLAKLGIKQDN 418

430 440 450 460 470 480 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc3p SVFKEFDPELRQQEEKHLHEAKLAKLEAEMKAVFQQKVSEKEKKLQKSEAELFARHKEMK 468 ScCdc3p SVFKEFDPISKQLEEKTLHEAKLAKLEIEMKTVFQQKVSEKEKKLQKSETELFARHKEMK 478

490 500 510 520 ....|....|....|....|....|....|....|....|.. AgCdc3p EKLMKQLKALEEKKHQLEMSL--ANQS-SQSPAQPKKKGFLR 507 ScCdc3p EKLTKQLKALEDKKKQLELSINSASPNVNHSPVPTKKKGFLR 520

Appendix 3.7. Alignment of AgCdc3p and ScCdc3p (1/1) 10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc10p MSSVADSSLITPSSYVGFDTITAQIEHRLLKRGFQFNIMVVGHSGLGKSTLINSLFASHL 60 ScCdc10p MDPL--SS-VQPASYVGFDTITNQIEHRLLKKGFQFNIMVVGQSGLGKSTLINTLFASHL 57

70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc10p IDSSTGKDITKEPITKTTEIKVSYHSLVEDKVRLNVNCIDTPGFGDQINNDKVWEPIVKY 120 ScCdc10p IDSATGDDISALPVTKTTEMKISTHTLVEDRVRLNINVIDTPGFGDFIDNSKAWEPIVKY 117

130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc10p IKEQHSQYLRKELTAQREKHIVDTRVHAVLYFIQPNGKGLTQLDIAALKRLTDITNVIPV 180 ScCdc10p IKEQHSQYLRKELTAQRERFITDTRVHAILYFLQPNGKELSRLDVEALKRLTEIANVIPV 177

190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc10p IAKADTLTMDERAKFREIIQHEFKKHNFRIYPYDSDDLTPEELELNDSIRSIIPFAVVGS 240 ScCdc10p IGKSDTLTLDERTEFRELIQNEFEKYNFKIYPYDSEELTDEELELNRSVRSIIPFAVVGS 237

250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc10p EKEITVNGEVVRGRKTRWGAINLEDINQCEFVYLREFLIRTHLQDLIETTALIHYESFRS 300 ScCdc10p ENEIEINGETFRGRKTRWSAINVEDINQCDFVYLREFLIRTHLQDLIETTSYIHYEGFRA 297

310 320 ....|....|....|....|....|... AgCdc10p KQLIALKENASSRATGHGAQSSSTNMLR 328 ScCdc10p RQLIALKENANSRSSAH---MSSNAIQR 322

Appendix 3.8. Alignment of AgCdc10p and ScCdc10p (1/1) 10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc11Ap MSSIIEASAALRKRKHLKRGIQFTLMVVGQSGSGRSTFINTLCGQEVVETSTTVMLPNDD 60 AgCdc11Bp MAGIIEASVALRKRKHLKRGIQFTVMVVGQSGSGRSTFINTLCGQEVVETSTTVLLPEDD 60 ScCdc11p MSGIIDASSALRKRKHLKRGITFTVMIVGQSGSGRSTFINTLCGQQVVDTSTTILLPTDT 60

70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc11Ap ATQIDVQLREETVELEDDEGVKIQLTIIDTPGFGDSLDNSPSFNMISDYIRHQYDEILLE 120 AgCdc11Bp AAQIDVQLREETVELEDDEGVKIQLTIIDTPGFGDSLYNSPSFNMISDYIRHQYDEILLE 120 ScCdc11p STEIDLQLREETVELEDDEGVKIQLNIIDTPGFGDSLDNSPSFEIISDYIRHQYDEILLE 120

130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc11Ap ESRVRRNPRFKDGRVHCCLYLINPTGHGLKEIDVEFMRQLGPLVNVIPVISKSDSLTPDE 180 AgCdc11Bp ESRVRRNPRFKDGRVHCCLYLINPTGHGLKEIDVEFMRQLGPLVNVIPVISKSDSLTPDE 180 ScCdc11p ESRVRRNPRFKDGRVHCCLYLINPTGHGLKEIDVEFIRQLGSLVNIIPVISKSDSLTRDE 180

190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc11Ap LKLNKKLIMEDIDYYNLPIYSFPFDQDVVSDEDYETNTYLRSLLPFSIIGSNETFET-AE 239 AgCdc11Bp LKLNKKLIMEDIDYYNLPIYSFPFDQDVVSDEDYETNTYLRSLLPFSIIGSNETFEA-AD 239 ScCdc11p LKLNKKLIMEDIDRWNLPIYNFPFDEDEISDEDYETNMYLRTLLPFAIIGSNEVYEMGGD 240

250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc11Ap GGVIHGRRYPWGTIDVEDPVVSDFCVLRNALLISHLNDLKDYTHELLYERYRTEALSGDM 299 AgCdc11Bp GRVIHGRRYPWGTVDVEDPVVSDFCVLRNALLISHLNDLKDYTHELLYERYRTEALSGDV 299 ScCdc11p VGTIRGRKYPWGILDVEDSSISDFVILRNALLISHLHDLKNYTHEILYERYRTEALSGES 300

310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc11Ap LTASSVSSKLMNNGSTEFISSPALSGTG--SDSARVSGQEAESRNSTK-QSS--NQDTYL 354 AgCdc11Bp LTASSVPSKTVINGSTEYAGSPVPPATA--SDSVHVSGHEAESRNSTK-QSS--NQDTYL 354 ScCdc11p VAAESIRPNLTKLNGSSSSSTTTRRNTNPFKQSNNINNDVLNPASDMHGQSTGENNETYM 360

370 380 390 400 410 ....|....|....|....|....|....|....|....|....|....|....|.. AgCdc11Ap AREEQIRLEEKRLKAFEERVQQELLSKRQELLRREQELREIEERLEKEAKTKQEIED 411 AgCdc11Bp AREEQLRLEEQRLKVFEERVQQELLSKRQELLRREQELREIEERLEKEAKTRVE--- 408 ScCdc11p TREEQIRLEEERLKAFEERVQQELLLKRQELLQREKELREIEARLEKEAKIKQEE-- 415

Appendix 3.9. Alignment of AgCdc11Ap, AgCdc11Bp and ScCdc11p (1/1) 10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc12p MLNRSD-----GSLVGISNLPNQRYKIVSNKGGVFTLMCCGESGLGKTTFINTLFQTTLS 55 ScCdc12p MSAATATAAPVPPPVGISNLPNQRYKIVNEEGGTFTVMLCGESGLGKTTFINTLFQTVLK 60

70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc12p PLENQNRRQQPIRKTAEVNVIRAMLEEKNFSLRVNVIDTPGFGDNVNNNKSWQTIIDFID 115 ScCdc12p RADGQQHRQEPIRKTVEIDITRALLEEKHFELRVNVIDTPGFGDNVNNNKAWQPLVDFID 120

130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc12p DQHDSYMRQEQQPYRSVKFDLRVHAVLYFIRPTGHGLKPLDIETMKRISTRANLIPVIAK 175 ScCdc12p DQHDSYMRQEQQPYRTKKFDLRVHAVLYFIRPTGHGLKPIDIETMKRLSTRANLIPVIAK 180

190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc12p ADTLTAKELQDFKVRIRQVIEAQDICIFTPPLDEADQD------DPAAMEHARQLVQS 227 ScCdc12p ADTLTAQELQQFKSRIRQVIEAQEIRIFTPPLDADSKEDAKSGSNPDSAAVEHARQLIEA 240

250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc12p MPFAVIGSEKKFDNGSGSLVAARKYPWGLVEVENDAHCDFRKLRSLLLRTNLLDLILTTE 287 ScCdc12p MPFAIVGSEKKFDNGQGTQVVARKYPWGLVEIENDSHCDFRKLRALLLRTYLLDLISTTQ 300

310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....| AgCdc12p ELHYETYRRLRLEGNSAAAE-EKDGTLPHPAPARKLSHNPKFKEEENALKKYFTDQVKAE 346 ScCdc12p EMHYETYRRLRLEGHENTGEGNEDFTLPAIAPARKLSHNPRYKEEENALKKYFTDQVKAE 360

370 380 390 400 ....|....|....|....|....|....|....|....|....|.. AgCdc12p EQRFRQWEQNIVSERIRLNGDLEEVQAKVKKLEEQVRALQLRKH--- 390 ScCdc12p EQRFRQWEQNIVNERIRLNGDLEEIQGKVKKLEEQVKSLQVKKSHLK 407

Appendix 3.10. Alignment of AgCdc12p and ScCdc12p (1/1) 10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| AgSep7p MTAVQYSNALPPALFRRKKEQKRGVTYSVLLVGPSGTGKTTFANNLLESTIFSHRYQ--- 57 ScSep7p ---MSTASTPPINLFRRKKEHKRGITYTMLLCGPAGTGKTAFANNLLETKIFPHKYQYGK 57

70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| AgSep7p TEQPQYQNPMVKVVNPTRVVTFNSKNGIPSYQVPFDPMGAHLEPGITITATSVEVSTDSS 117 ScSep7p SNASISSNPEVKVIAPTKVVSFNSKNGIPSYVSEFDPMRANLEPGITITSTSLELGGNKD 117

130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....| AgSep7p SDDP----RDKMCFNLIDTHGIGENLDNELCFDEVVAYLEQQFDLVLAEETRIKRNPRFE 173 ScSep7p QGKPEMNEDDTVFFNLIMTHGIGENLDDSLCSEEVMSYLEQQFDIVLAEETRIKRNPRFE 177

190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....| AgSep7p DGRIHAALYFVEPTGHGLRELDIEMMKRLSRYTNVLPIIARADSFMEDELSSFKAAVMRD 233 ScSep7p DTRVHVALYFIEPTGHGLREVDVELMKSISKYTNVLPIITRADSFTKEELTQFRKNIMFD 237

250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....| AgSep7p IENYNVPVYKFEVDEDEDDPETLQEVGDLAAIQPFAVVCSDTKGKDGRYVRAYPWGDLFI 293 ScSep7p VERYNVPIYKFEVDPEDDDLESMEENQALASLQPFAIITSDTRDSEGRYVREYPWGIISI 297

310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....| AgSep7p DDETVSDLRVLKSVLFGSYLQEFKDTTHNLLYENYRAEKLSSISEWDTTKTSSKGTSIVK 353 ScSep7p DDDKISDLKVLKNVLFGSHLQEFKDTTQNLLYENYRSEKLSSV------ANAEEIGPNS 350

370 380 390 400 410 420 ....|....|....|....|....|....|....|....|....|....|....|....| AgSep7p TEKNSSTPSLSNFASIVNTGNLKSQQSLTKVPNSDVPAPSTPSNESDSLF------403 ScSep7p TKRQSNAPSLSNFASLISTGQFNSSQTLANNLRADTPRNQVSGNFKENEYEDNGEHDSAE 410

430 440 450 460 470 480 ....|....|....|....|....|....|....|....|....|....|....|....| AgSep7p RETE-SPIRKMSVNIRRDNEEIIRNIK--SSPST-AESGSQDRTKLRNISETLPYVLRHE 459 ScSep7p NEQEMSPVRQLGREIKQENENLIRSIKTESSPKFLNSPDLPERTKLRNISETVPYVLRHE 470

490 500 510 520 530 540 ....|....|....|....|....|....|....|....|....|....|....|....| AgSep7p RIIAKQQKLEELEAQSARELQKRIQELEKKAMELKLKEKLLKQQKRNGSTTSIGSTVTAT 519 ScSep7p RILARQQKLEELEAQSAKELQKRIQELERKAHELKLREKLINQNKLNGSSSSI------523

550 560 570 580 590 600 ....|....|....|....|....|....|....|....|....|....|....|....| AgSep7p SAASTNLTSGKPTIMQEGTPSTVNRARNGSLVSPSTQVSPSADSSQFASDGYASRATGQV 579 ScSep7p ------NS--LQQ---ST------RS-QIKK---NDTY-T-DLASIASGR- 550

. AgSep7p Q 580 ScSep7p D 551

Appendix 3.11. Alignment of AgSep7p and ScSep7p (1/1) References

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REFERENCES 47 Acknowledgements

I am very grateful to Prof. Peter Philippsen for having accepted me as a member of his team, for guiding me through all these years with valuable scientific and personal advises and for making this work possible through his generous support. His ability to keep up a warm and familial atmosphere in the team not only made this time rich in experience and education, but also made it a very enjoyable part of my life. “Thesis supervisor” does not describe Peter’s role adequately enough; I prefer “Doktorvater”.

Similarly, I would like to thank my “Doktormutter”, Dr. Amy Gladfelter for lending her time, her experience, ideas, and moral support. She always had an open ear for my thoughts – no matter how crazy they were, and got never tired to comfort and encourage me – in scientific and personal matters.

I also wish to acknowledge the other members of my thesis committee and participants of my exam, Prof. Yves Barral, Prof. Mike Hall, Prof. Anne Spang and Prof. Jean Pieters for lending their time and offering helpful suggestions on my project.

My warm thanks to all present and former members of the “lab family”, especially Philippe Laissue for his loyal friendship and for sharing my peculiar taste of humor, Michael Köhli for accompanying me through the odyssey in Copenhagen and for sharing adventures with “Bonny and Clyde”, Sylvia Vögeli, Virginie Galati for making lunch times and coffee breaks amusing events, Riccarda Rischatsch, Philipp Knechtle, Anita Lerch, Sophie Brachat and Daniele Cavicchioli for having been wonderful office mates, Kamila Boudier for encouraging discussions and memorable parties, Dominic Hœpfner, Hans- Peter Schmitz, Florian Schärer, Christine Alberti-Segui and Yasmina Bauer for teaching me first and advanced steps in lab techniques, Andreas Kaufmann for his unfailing engagement in keeping lab equipment running, Claudia Birrer, Nicoleta Sustreanu, Tineke van den Hoorn, Pilar Garcia, Kathrin Brogli, Ivan Schlatter, Nijas Alijoski, Jérôme Ailhas, Zaki Sellam, and the new diploma students for adding to the friendly atmosphere in the lab, Brigitte Berglas for constantly providing the lab with perfectly clean vessels, and Bettina Hersberger, Sandra Götz-Krebs, Doris Rossi and Esther Graf for their help concerning office and paperwork.

I additionally thank my colleagues and friends from the Biozentrum, Petr Broz, Thomas Rhomberg and Stefan Langheld, for sharing joy and sorrow over a drink or at the movies, and my trusty friend Cyrill Rémy for the inspiring and philosophical discussions about biology and other matters of life.

None of this would have possible without the unwavering support and love of my parents, Madeleine and Fritz Helfer. Especially, I would like to thank my dear Katrin for sweetening my life since the last year of my thesis work, for her unfailing support and patience, for buoying me up in desperate moments, and most of all: for her love.

ACKNOWLEDGEMENTS 48 Curriculum vitae

Hanspeter Helfer

Date of birth: 11.10.1976 Place of birth: Selzach, Switzerland Nationality: Swiss

Education:

Primary school: 1983 – 1989 Primarschule Selzach, Switzerland

Secondary school: 1989 – 1992 Bezirksschule Selzach, Switzerland

Grammar school: 1992 – 1997 Kantonsschule Solothurn, Switzerland

University: 1997 – 2002 Diploma in Biology II (Molecular Biology), Universität Basel, Switzerland

Diploma Thesis: 2000 – 2002 In the lab of Prof. P. Philippsen, Biozentrum, Basel, Switzerland “Effects of myosin gene deletions on growth and development of the filamentous fungus Ashbya gossypii”

Ph.D.: 2002 – 2006 In the lab of Prof. P. Philippsen, Biozentrum, Basel, Switzerland “New aspects of septin assembly and cell cycle control in multinucleated A. gossypii”

Publications

H Helfer and AS Gladfelter. AgSwe1p regulates mitosis in response to morphogenesis and nutrients in multinucleated A. gossypii cells. MBC, selected for InCytes, Epub ahead of print August 9, 2006.

Y Bauer, P Knechtle, J Wendland, H Helfer and P Philippsen. A Ras-like GTPase is involved in hyphal growth guidance in the filamentous fungus Ashbya gossypii. MBC, Vol. 15, Issue 10, 4622-4632, October 2004

CURRICULUM VITAE 49 Meetings and Seminars

European Conference on Fungal Genetics, Pisa, Italy, April 2002. Poster presentation: Effects of myosin gene deletions on growth and development of the filamentous fungus Ashbya gossypii.

Swiss Yeast Meeting, Zürich, Switzerland. September 2002. Poster presentation: Effects of myosin gene deletions on growth and development of the filamentous fungus Ashbya gossypii.

European Conference on Fungal Genetics, Copenhagen, Denmark, April 2004. Oral presentation: In search of a link between morphogenesis and nuclear division in the filamentous fungus Ashyba gossypii.

Fungal Genetics Conference, Asilomar, CA, USA, March 2005. Poster presentation: Spatial and nutritional control of mitosis in Ashyba gossypii.

CURRICULUM VITAE 50 Erklärung

Ich erkläre, dass ich die Dissertation

“New aspects of septin assembly and cell cycle control in multinucleated A. gossypii” nur mit der darin angegebenen Hilfe verfasst und bei keiner anderen Fakultät eingereicht habe.

Basel, Mai 2006

Hanspeter Helfer

ERKLÄRUNG 51